Peritoneal dialysis-related peritonitis (PDRP) is a major complication of home-based dialysis, often leading to catheter failure, hospitalization, or mortality. Current diagnostic approaches such as culture and Gram staining are slow, labor-intensive, and prone to false negatives. Here we report a culture-free triplex fluorescence aptasensor for the simultaneous detection of Staphylococcus aureus and Pseudomonas aeruginosa, two key pathogens in PDRP. The sensor design is based on a triplex DNA structure comprising an unlabelled aptamer flanked by fluorophore- and quencher-labeled complementary strands. In the presence of target bacteria, aptamer binding disrupts the triplex, leading to fluorescence restoration. The aptasensors demonstrated excellent sensitivity, high specificity against non-target organisms, and robust performance in sterilized clinical peritoneal fluid. This simple, cost-effective assay shows strong potential for rapid point-of-care diagnostics in peritoneal dialysis, enabling rapid identification of pathogens and timely clinical decision-making.