AIM: To estimate if nanopore targeted sequencing (NTS) could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS. METHODS: A total of 55 patients (55 eyes) with postoperative endophthalmitis were retrospectively included in this study with their medical records. Intraocular fluid samples were examined by NTS and microbial culture. All included patients had undergone examinations including measurement of best corrected visual acuity (BCVA) and intraocular pressure (IOP), slit-lamp biomicroscopy, and indirect ophthalmoscopy; additionally, they underwent B-ultrasound, anterior segment photography, and fundus photography if necessary. RESULTS: Among 55 patients with postoperative endophthalmitis, the age was 65.25±15.04y and there were 30 female (54.54%) patients. Forty-one (74.54%) vitreous humor samples and fourteen (25.45%) aqueous humor samples were sent for both NTS and microbial culture. NTS had a notable higher detection rate than microbial culture in detecting pathogens (90.91% vs 38.18%, χ2=33.409, P<0.001). NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples (100% and 85.29%, respectively). In 16 of 21 (76.19%) patients who showed culture-positivity, their results corresponded with those of NTS. Moreover, in two patients (9.52%), NTS showed a better species resolution than microbial culture; in three patients (14.28%), NTS identified additional pathogens. As for fungus, the positive detection rate of NTS was significantly higher than that of microbial culture (20% vs 3.64%, χ2=7.066, P=0.008). Also, NTS could detect multi-infection by bacteria and fungi than microbial culture (32.73% vs 0, χ2=21.522, P<0.001). NTS could detect bacteria as well as fungi simultaneously within 48h in all patients. Meanwhile, NTS had a shorter detection time than microbial culture (1.13±0.34 vs 2.67±0.55d, Z= -9.218, P<0.001). After the NTS results were obtained, 15 patients received additional intravitreal/intracameral anti-infection treatment. At follow-up, there was a statistically significant improvement in the visual acuity relative to the baseline (Z=−5.222, P<0.001). CONCLUSION: NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis, which can guide anti-infection treatment and improve visual prognosis.