Objective To investigate the independent predictive factors for functional cure after long-term nucleos(t)ide analogue (NUC) antiviral therapy followed by pegylated interferon α-2b therapy in chronic hepatitis B (CHB) patients. Methods A total of 162 CHB patients who were admitted to several hospitals in Qingdao, China, from 2018 to 2021 were enrolled as subjects, and all patients received pegylated interferon α-2b for at least 48 weeks after NUC therapy for one year or longer. According to whether HBsAg clearance was achieved at week 48 of pegylated interferon α-2b treatment, the patients were divided into functional cure group with 79 patients and non-cure group with 83 patients, and related clinical indices were compared between the two groups. The two-independent-samples t test and the Mann-Whitney U rank sum test were used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups. The Spearman correlation analysis was performed, and the univariate and multivariate logistic regression analyses were used to investigate the independent predictive factors for functional cure. The receiver operating characteristic (ROC) curve was plotted for related variables, and the area under the ROC curve (AUC) was used to evaluate the prediction accuracy of the variables. Results Compared with the non-cure group, the functional cure group had a significantly lower HBsAg level at baseline [21.63 (3.33-157.60) IU/mL vs 794.70 (336.10-1 185.34) IU/mL, Z =-8.869, P < 0.001], at week 12 of pegylated interferon α-2b treatment [1.34 (0.04-16.59) IU/mL vs 567.11 (226.09-1 047.86) IU/mL, Z =-9.847, P < 0.001), and at week 24 of pegylated interferon α-2b treatment [0.01 (0.00-0.34) IU/mL vs 304.79 (89.24-772.23) IU/mL, Z =-10.474, P < 0.001) and a significantly greater reduction in HBsAg at weeks 12 and 24 of pegylated interferon α-2b treatment [week 12: 89.6% (57.5%-99.4%) vs 21.8% (2.0%-40.9%), Z =-7.926, P < 0.001; week 24: 99.9% (99.0%-100.0%) vs 44.1% (20.6%-73.8%), Z =-9.593, P < 0.001]. Compared with the non-cure group, the functional cure group had a significantly lower HBeAg positive rate at baseline (8.9% vs 25.3%, χ 2 =7.652, P =0.006), a significantly lower proportion of patients with baseline HBV DNA > 1000 IU/mL (0 vs 8.4%, χ 2 =5.073, P =0.024), a significantly lower level of total bilirubin at baseline [12.60 (10.12-15.93) μmol/L vs 15.50 (11.80-24.10) μmol/L, Z =-3.611, P < 0.001], a significantly higher level of aspartate aminotransferase (AST) at week 12 of treatment [47.00 (34.00-68.00) U/L vs 41.00 (30.00-56.50) U/L, Z =-2.031, P =0.042], and a significantly higher proportion of patients with AST > 2×upper limit of normal (16.5% vs 4.8%, χ 2 =5.835, P =0.016). The multivariate logistic regression analysis showed that baseline HBsAg (odds ratio [ OR ]=0.996, 95% confidence interval [ CI ]: 0.995-0.997, P < 0.001), HBsAg at week 12 of pegylated interferon α-2b treatment ( OR =0.990, 95% CI : 0.986-0.994, P < 0.001), HBsAg at week 24 of pegylated interferon α-2b treatment ( OR =0.983, 95% CI : 0.975-0.991, P < 0.001), and baseline total bilirubin ( OR =0.885, 95% CI : 0.826-0.949, P =0.001) were independent predictive factors for functional cure. The ROC curve of baseline HBsAg showed an AUC of 0.904 and the optimal cut-off value of 118.24 IU/mL; the ROC curve of HBsAg at week 12 of pegylated interferon α-2b treatment showed an AUC of 0.948 and the optimal cut-off value of 73.74 IU/mL; the ROC curve of HBsAg at week 24 of pegylated interferon α-2b treatment showed an AUC of 0.975 and the optimal cut-off value of 11.01 IU/mL; the ROC curve of baseline total bilirubin showed an AUC of 0.664 and the optimal cut-off value of 19.9 μmol/L. Conclusion Baseline HBsAg, HBsAg at week 12 of pegylated interferon α-2b treatment, HBsAg at week 24 of pegylated interferon α-2b, and baseline total bilirubin are independent predictive factors for functional cure at week 48 of pegylated interferon α-2b treatment in CHB patients receiving sequential therapy with NUC and pegylated interferon α-2b.