Objective To explore the accuracy of fluid responsiveness assessment by variability of peripheral arterial peak velocity and variability of inferior vena cava diameter (ΔIVC) in patients with septic shock. Methods A prospective study was conducted. The patients with septic shock undergoing mechanical ventilation (MV) admitted to intensive care unit (ICU) of Beijing Electric Power Hospital from January 2016 to December 2017 were enrolled. According to sepsis bundles of septic shock, volume expansion (VE) was conducted. The increase in cardiac index (ΔCI) after VE ≥ 10% was defined as liquid reaction positive (responsive group), ΔCI < 10% was defined as the liquid reaction negative (non-responsive group). The hemodynamic parameters [central venous pressure (CVP), intrathoracic blood volume index (ITBVI), stroke volume variation (SVV), ΔIVC, variability of carotid Doppler peak velocity (ΔCDPV), and variability of brachial artery peak velocity (ΔVpeak-BA)] before and after VE were monitored. The correlations between the hemodynamic parameters and ΔCI were explored by Pearson correlation analysis. Receiver operating characteristic (ROC) curve was plotted to analyze the predictive value of all hemodynamic parameters on fluid responsiveness. Results During the study, 74 patients with septic shock were included, of whom 9 were excluded because of peripheral artery stenosis, recurrent arrhythmia or abdominal distension influencing the ultrasound examination, and 65 patients were finally enrolled in the analysis. There were 31 patients in the responsive group and 34 in the non-responsive group. SVV, ΔIVC, ΔCDPV and ΔVpeak-BA before VE in responsive group were significantly higher than those of the non-responsive group [SVV: (12.3±2.4)% vs. (9.2±2.1)%, ΔIVC: (22.3±5.3)% vs. (15.5±3.7)%, ΔCDPV: (15.3±3.3)% vs. (10.3±2.4)%, ΔVpeak-BA: (14.5±3.3)% vs. (9.6±2.3)%, all P < 0.05]. There was no significant difference in CVP [mmHg (1 mmHg = 0.133 kPa): 7.5±2.5 vs. 8.2±2.6] or ITBVI (mL/m2: 875.2±173.2 vs. 853.2±192.0) between the responsive group and non-responsive group (both P > 0.05). There was no significant difference in hemodynamic parameter after VE between the two groups. Correlation analysis showed that SVV, ΔIVC, ΔCDPV, and ΔVpeak-BA before VE showed significant linearity correlation with ΔCI (r value was 0.832, 0.813, 0.854, and 0.814, respectively, all P < 0.05), but no correlation was found between CVP and ΔCI (r = -0.342, P > 0.05) as well as ITBVI and ΔCI (r = -0.338, P > 0.05). ROC curve analysis showed that the area under ROC curve (AUC) of SVV, ΔIVC, ΔCDPV, and ΔVpeak-BA before VE for predicting fluid responsiveness was 0.857, 0.826, 0.906, and 0.866, respectively, which was significantly higher than that of CVP (AUC = 0.611) and ITBVI (AUC = 0.679). When the optimal cut-off value of SVV for predicting fluid responsiveness was 11.5%, the sensitivity was 70.4%, and the specificity was 94.7%. When the optimal cut-off value of ΔIVC was 20.5%, the sensitivity was 60.3%, and the specificity was 89.7%. When the optimal cut-off value of ΔCDPV was 13.0%, the sensitivity was 75.2%, and the specificity was 94.9%. When the optimal cut-off value of ΔVpeak-BA was 12.7%, the sensitivity was 64.8%, and the specificity was 89.7%. Conclusions Ultrasound assessment of ΔIVC, ΔCDPV, and ΔVpeak-BA could predict fluid responsiveness in patients with septic shock receiving mechanical ventilation. ΔCDPV had the highest predictive value among these parameters.