To enhance the accuracy and efficiency of pre-transfusion irregular antibody screening, this study integrates microfluidic chip technology with transfusion screening requirements. We designed an integrated screening chip and optimized the reaction system and operational workflow. Performance validation demonstrated that the detection limits for anti-E and anti-Le antibodies were ≤ 0.5 IU/mL, ≤ 0.8 IU/mL, with specificity ≥ 98%. The single-sample testing cycle was completed within 30 minutes using only 10 μL of sample. Clinical testing showed a 3.12% antibody detection rate in patients undergoing multiple transfusions, achieving 99.8% consistency with conventional blood bank methods. Post-application cross-matching missed detection rate was ≤ 0.1%, and transfusion adverse reactions decreased by 30%, providing efficient technical support for clinical transfusion safety with promising application prospects. As a cutting-edge interdisciplinary technology integrating microelectronics, fluid mechanics, chemistry, and biology, microfluidic chip technology has experienced rapid development over the past decades. It has demonstrated significant application potential in medical diagnostics, biological research, and chemical analysis, emerging as one of the most prominent research hotspots in both scientific and industrial fields.