Purpose This paper aimed to assess the diagnostic utility of a newly developed gene-based technology-nanopore targeted sequencing (NTS) in suspected endophthalmitis patients. Methods This retrospective study included 43 patients (44 eyes) with suspected endophthalmitis. NTS was applied along with microbiological culture to detect unknown pathogens in intraocular fluid samples. The diagnostic utility of NTS was mainly evaluated from three aspects, including the positivity rate of bacterial/fungal presence, diagnostic turnaround time and the frequency of change in treatment based on etiology test results. Non-parametric, two-sided Wilcoxon rank sum test, the McNemar’s test and the kappa statistic were used for statistical comparisons. Results NTS showed significant advantages over traditional culture in positivity rates and diagnostic time (P < 0.001, kappa = 0.082; Z = −5.805, P < 0. 001). As regards antibiotic strategy, 17 patients (39.53%) and 5 patients (11.63%) underwent medication change following NTS and culture results respectively (P < 0.001, kappa = 0.335). With reasonable use of antibiotic and surgical intervention, most patients responded favorably, judged by significantly improved visual acuity (Z = −4.249, P < 0.001). The mean duration of hospitalization was 8.49 ± 2.45 days (range, 1–16 days). Conclusion The high efficiency feature of NTS in pathogen detection renders it a valuable supplementary to traditional culture. Additionally, it has facilitated patients’ management for the early and precise diagnosis of endophthalmitis.