Abstract Background Surface antigens on platelets and neutrophils—including human platelet antigens (HPA), human neutrophil antigens (HNA), and CD36—are critical determinants of compatibility in platelet transfusion. To address the need for high‐throughput genotyping, this study developed a method for simultaneous detection of 35 HPA systems, 5 HNA systems, and CD36 polymorphisms based on matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) technology. Study Design and Methods Specific primers for amplification and single‐base extension were designed, distributing 53 loci into two multiplex pools. Genotyping was performed using the MassARRAY MS platform. Method accuracy was verified by Sanger sequencing for all 53 loci (34 samples) and flow cytometric serotyping for HPA‐1, ‐3, ‐5 and CD36. The validated method was then applied to genotype 8100 random blood donors to establish a platelet donor database. Results An MS‐based genotyping method was successfully established, covering 53 loci distributed across two multiplex pools. All verified samples yielded results consistent with those obtained through Sanger sequencing, serotyping, or flow cytometry. Using this method, a platelet donor genotyping database comprising 8100 donors was created, identifying rare HPA alleles (1 case each of HPA‐8b/‐9b/‐11b/‐16b/‐25b; 2 of HPA‐17b/‐30b; 3 of HPA‐20b). Discussion We successfully developed an MS panel capable of detecting all currently known HPAs, HNAs, and the CD36 antigen, overcoming limitations of previous methods related to non‐specific HNA‐1 amplification. This method also demonstrated its efficacy in establishing a large‐scale platelet donor database, and rare allele data support precise transfusion matching for special patients.