Objective To investigate the specific distribution of weakly positive irregular antibody screening results, their underlying causes, and their impact on clinical transfusion safety, thereby providing evidence for optimizing laboratory review procedures and clinical management strategies for weakly positive specimens. Methods A retrospective study was conducted by collecting data from 186 patients with weakly positive irregular antibody screening results identified at the Blood Group Laboratory of Xuchang Central Blood Station between December 1, 2022, and December 31, 2024. All specimens were screened using the microcolumn gel anti-human globulin method. Weakly positive samples underwent further antibody identification and verification using the saline method and anti-human globulin tube test. Combined with patient transfusion histories and clinical diagnoses, we analyzed the distribution characteristics and associated factors of weakly positive antibodies. Results Among the 186 weakly positive samples, 89 (47.8%) were male and 97 (52.2%) were female; 68.8% had a history of transfusion. Antibody identification revealed that 76.3% (142/186) were antibodies with clear specificity, predominantly within the Rh system (37.3%), MNSs system (21.8%), and P system (16.2%), with common antibodies including anti-E, anti-P1, and anti-M. The saline test detected only 62.7% (89/142) of specific antibodies, suggesting a predominance of IgG class. Patients with multiple transfusions (≥3 times) exhibited significantly higher weak positive rates than those with fewer transfusions (P＜0.001). No acute hemolytic reactions occurred after transfusion of antigen-negative blood, but 13.8% of patients receiving incompatible blood experienced transfusion failure. Conclusion Weakly positive irregular antibody screening results are largely clinically significant and closely correlated with transfusion history. Such antibodies commonly belong to the Rh system and are predominantly IgG-type, often missed by saline testing. Clinically, a systematic re-evaluation process for weakly positive results should be established, employing multiple methods for confirmation. Patients with positive specific antibodies should receive antigen-negative blood transfusions. Enhanced screening and follow-up for multiple transfusion recipients are essential to improve transfusion safety and management effectiveness.