Objective This study aims to analyze clinical data collected from patients with positive accidental antibody screening results, exploring the distribution of accidental antibodies and their correlation with transfusion history and pregnancy history, providing a reference basis for ensuring clinical transfusion safety. Methods This research collected clinical data from patients with positive and negative accidental antibody screening results at Xiangya Third Hospital of Central South University from April 2020 to March 2021, and conducted a retrospective analysis. Results Among the 116 patients, 66 cases of accidental antibody positivity were identified after excluding incomplete data, including 19 males and 47 females. Accidental antibodies were mainly distributed in the Rh system, with anti-E being the most common, accounting for 30.3%; the Lewis blood group system accounted for 18.18%, with some patients exhibiting both anti-Lea and anti-Le^b; in the MNS blood group system, anti-M accounted for 13.64% and anti-Mur antibodies accounted for 9.09%. Additionally, autoantibodies accounted for 3.03%, mixed antibodies accounted for 1.52%, and specific antibodies not detected accounted for 1.52%. Gender, transfusion history, and pregnancy history were statistically significant factors in the detection rate of accidental antibodies. Furthermore, multivariable logistic regression analysis indicated that a history of transfusion and pregnancy were independent risk factors for the production of accidental antibodies. Conclusion The detection rate of anti-E in the Rh blood group system is the highest, which requires attention. It is recommended to establish a Rh blood group system donor phenotype database to provide safer and faster transfusion treatment for patients.