Introduction To evaluate insulin injection knowledge, attitudes, and practices of nurses across China in order to provide reference for the formulation of a national unified standard of insulin injection practice and the targeted implementation of standardized training on insulin injection for nurses. Methods We enrolled nurses who worked and injected insulin at grassroot hospitals including community health service centers and township clinics, secondary and tertiary care hospitals across China between July 28, 2019 and August 30, 2019. A nurse insulin injection knowledge, attitude, and practice questionnaire was used to evaluate the knowledge, attitude, and practice level of nurses. Results A total of 223,368 nurses were included in the study. The mean knowledge score was 13.70 +/- 3.30 and 35.19% had a poor knowledge score. The mean attitude score was 17.18 +/- 2.69 for the study nurses; merely 3.15% had a poor attitude score. The mean practice score of the study population was 83.03 +/- 8.16 and only 0.88% had a poor practice score. Pearson correlation analysis showed significant correlation between the knowledge score and the attitude score (r = 0.29; P < 0.001), the knowledge score and the practice score (r = 0.27; P < 0.001), and between the attitude score and the practice score (r = 0.56; P < 0.001). A multivariate analysis revealed that nurses with higher knowledge scores were also more likely to have higher attitude scores and practice scores, and nurses with higher attitude scores were also more likely to have higher practice scores. Conclusion Chinese nurses have a good attitude and behavior towards insulin injection, while their knowledge of insulin injection is insufficient. It is also revealed that knowledge of insulin injection can directly or indirectly affect insulin injection behavior through attitude, indicating that hospitals should formulate unified insulin injection norms and regularly organize relevant training and assessment so as to improve nurses' knowledge, attitude, and behavior of insulin injection.