Prompt clinical diagnosis and antimicrobial therapy are key to managing infective endophthalmitis. The small volume of aqueous humor, low bacterial counts, and empirical medication by physicians make existing diagnostic methods time-consuming and imprecise. Here, we investigated the feasibility of combining Fc-MBL@Fe3O4 enrichment with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling to identify pathogens in aqueous humor. Aqueous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), and Klebsiella pneumoniae (K. pneumoniae). We performed identification directly in aqueous humor samples and after short-term culture of micro-LB broth. Aqueous humor endophthalmitis samples were enriched with Fc-MBL@Fe3O4 and analyzed with MALDI-TOF MS. The identification time and minimum bacterial concentration required for identification were determined. The enrichment efficiency of Fc-MBL@Fe3O4 for different bacteria was > (87.5±5.0)%. The objects of direct identification include live bacteria and bacteria treated with antibiotics, which can be completed within 1.5 hours. The minimum number of bacteria needed for positive identification was 2.20×106 CFU. For micro-LB broth culture, the identification of bacteria can be completed within 6.5-9.5 h for aqueous humor samples with an initial bacterial count of tens to hundreds.