Chemiluminescence (CL) with stable and glowing light emission is vital for the accurate detection of biomarkers. Moreover, the catalyst plays an important role in CL systems. Herein, the trimetallic AuPtCo nanopolyhedrons with peroxidase- and catalase-like catalytic activities are readily synthesized via a one-step reduction method. After reaction with the substrate ABEI and oxidant H2O2, the AuPtCo nanozyme can catalyze the CL emission in a flash type. Interestingly, it has been found that the biofunctionalization of the AuPtCo surface can endow the catalytic interface with a slow-diffusion effect, thereby prolonging the emission of glow-type CL. On this basis, two biofunctionalized AuPtCo nanocomposites, named as AuPtCo@Cys and AuPtCo@Ab, are prepared, achieving sensitive and selective detection of H2O2 and lipoprotein-associated phospholipase A2 (Lp-PLA2), respectively. Further, the proposed glow-type CL assays are successfully applied for the determination of H2O2 and Lp-PLA2 in female vaginal discharge and human serum samples, respectively, which exhibit good correlation with the clinical results. Overall, the trimetallic AuPtCo nanozyme-based glow-type CL analysis has demonstrated as a powerful and robust tool for biomarker analysis, which holds great promise in clinical applications.