In addition to technical barriers, public attitudes about the use of gene therapy have an important association with the clinical implementation of gene therapy. To investigate the factors associated with public acceptance of gene therapy among individuals in China. This cross-sectional study used data from a survey conducted among 21 880 individuals in mainland China from June 20 to August 31, 2022. Stepwise linear regression was used to analyze factors associated with public acceptance of gene therapy in 5 key areas: basic personal information (gender, region, age, and educational level), family situation (marital status, children, and cousins), economic status (assets, debts, and insurance coverage), health knowledge (health literacy score and media use), and physical health status (chronic illness, cancer, European Quality of Life 5-Dimension 5-Level version [EQ-5D-5L] score, and Brief Illness Perception Questionnaire [BIPQ] score). Acceptance scores were calculated based on a visual analog scale (range, 0-100, with higher scores indicating higher acceptance of gene therapy). Further subgroup analysis was carried out in different age subgroups and populations with or without chronic diseases. A total of 21 880 participants (mean [SD] age, 39.4 [18.9] years; 10 947 female participants [50.0%]; 10 933 male participants [50.0%]) were analyzed in this study. The mean (SD) acceptance score of gene therapy in the survey was 60.56 (27.60). Compared with people aged 60 years or older, those aged 12 to 18 years had higher acceptance of gene therapy (β = 1.48 [95% CI, 0.09-2.88]), while groups aged 19 to 30 years (β = -3.43 [95% CI, -4.80 to -2.07]), 31 to 44 years (β = -1.44 [95% CI, -2.76 to -0.12]), and 45 to 59 years (β = -2.05 [95% CI, -3.27 to -0.83]) had lower acceptance. Compared with people living in Eastern China, those in Central China had lower acceptance of gene therapy (β = -1.58 [95% CI, -2.54 to -0.62]), while those in Western China had higher acceptance (β = 0.92 [95% CI, 0.09-1.76]). Higher educational level (undergraduate or above vs junior high or below) was associated with higher acceptance of gene therapy (β = 1.56 [95% CI, 0.49-2.63]). Number of properties owned was also associated with higher acceptance of gene therapy (2 vs 0: β = 2.38 [95% CI, 1.04-3.72]; ≥3 vs 0: β = 4.66 [95% CI, 2.92-6.39]). Diagnosis of chronic disease was associated with lower acceptance of gene therapy (β = -17.86 [95% CI, -20.49 to -15.24]), while diagnosis of cancer was associated with higher acceptance (β = 6.99 [95% CI, 1.84-12.14]). Higher BIPQ score (β = 0.40 [95% CI, 0.34-0.45]), higher health literacy score (β = 0.70 [95% CI, 0.62-0.78]), and media use (β = 0.49 [95% CI, 0.41-0.57]) were all associated with high acceptance of gene therapy, while a higher EQ-5D-5L score was associated with lower acceptance (β = -0.29 [95% CI, -0.47 to -0.11]). For older people, being in debt, not having health insurance, and the EQ-5D-5L score were uniquely relevant factors. For people with chronic disease, having an undergraduate degree or higher, a diagnosis of cancer, and the BIPQ score were uniquely relevant factors. These results suggest that basic personal information, economic status, health knowledge, and physical health status were the main factors associated with the acceptance of gene therapy. Improving the health literacy of the population and promoting trust in gene therapy may be effective ways to increase the acceptance of gene therapy. Poorer economic levels and worse disease states may reduce the public's willingness to accept gene therapy.