Background Cumulative fatigue without intervention will seriously threaten the physical and mental health of workers. Shift work and life satisfaction are strongly associated with fatigue accumulation. Objective To explore the effects of life satisfaction, shift work, and their interaction on cumulative fatigue in petrochemical employees, and to provide a scientific basis for preventing cumulative fatigue. Methods All staff of a petrochemical enterprise were selected by cluster sampling for a cross-sectional study from July to October 2021 in Jiangsu Province. A questionnaire designed by the project team was used to collect information on shift work; and life satisfaction and cumulative fatigue were investigated by the World Health Organization Five-item Well-Being Index and the Self-diagnosis Checklist for Assessment of Worker’s Fatigue Accumulation respectively. A logistic regression model was used to analyze the influences of life satisfaction and shift work on cumulative fatigue. Multiplicative and additive models were applied to analyze the interaction effect of life satisfaction and shift work. Results A total of 4066 questionnaires were returned, of which 3763 were valid, with an effective recovery rate of 92.5%. The percentage of cumulative fatigue in the petrochemical employees was 63.2% (2377/3763), and the percentages of low life satisfaction and shift work in the petrochemical employees were 53.6% (2016/3763) and 54.2% (2041/3763), respectively. The results of univariate analysis showed no significant difference in cumulative fatigue among different marital status groups (P=0.176), and there were statistically significant differences in cumulative fatigue among the petrochemical employees in different groups of age, gender, educational level, average monthly income, job title, length of service, working hours, night shift, smoking, drinking, physical exercise, life satisfaction, and shift work (P<0.001). After adjustment for covariates such as age, gender, educational level, average monthly income, job title, length of service, working hours, night shift, smoking, drinking, and physical activity, the unconditional logistic regression model showed that the risk of reporting cumulative fatigue in high life satisfaction participants was 0.129 (95%CI: 0.109, 0.154) times of that in participants of low life satisfaction; the risk of reporting cumulative fatigue in shift work participants was 3.792 (95%CI: 2.713, 5.300) times of that in no shift work participants; and the risk of reporting cumulative fatigue in participants with both high life satisfaction and shift work was 0.105 (95%CI: 0.081, 0.135) times of that in participants with low life satisfaction and shift work. The relative excess risk due to interaction, the attributable proportion due to interaction, and the synergy index of coexisting life satisfaction and shift work were −5.504 (95%CI: −7.247, −3.760), −4.728 (95%CI: −7.575, −1.880), and 0.029 (95%CI: 0.002, 0.351) respectively, which suggested that life satisfaction and shift work have an additive interaction effect on cumulative fatigue. A significant multiplicative interaction was also found between life satisfaction and shift work (OR=0.688, 95%CI: 0.476, 0.936). Conclusion Life satisfaction and shift work are the influencing factors of cumulative fatigue among petrochemical employees, and they interact with each other on the risk of cumulative fatigue. High life satisfaction can reduce the risk of accumulated fatigue associated with shift work.