Cancer patients are at high risk of infections and infection-related mortality; thereby, prompt diagnosis and precise anti-infectives treatment are critical. This study aimed to evaluate the performance of nanopore amplicon sequencing in identifying microbial agents among immunocompromised cancer patients with suspected infections. This prospective study enlisted 56 immunocompromised cancer patients with suspected infections. Their body fluid samples such as sputum and blood were collected, and potential microbial agents were detected in parallel by nanopore amplicon sequencing and the conventional culture method. Among the 56 body fluid samples, 47 (83.9%) samples were identified to have at least one pathogen by nanopore amplicon sequencing, but only 25 (44.6%) samples exhibited a positive finding by culture. Among 31 culture-negative samples, nanopore amplicon sequencing successfully detected pathogens in 22 samples (71.0%). Nanopore amplicon sequencing showed a higher sensitivity in pathogen detection than that of the conventional culture method (83.9% vs. 44.6%, P