Objective To investigate the application value of metagenomic next-generation sequencing (mNGS) in leukemia children with febrile neutropenic (FN). Methods The clinical data of leukemia children combined with FN after chemotherapy admitted to the pediatric intensive care unit from January 1, 2018 to July 1, 2021 was retrospectively analyzed. The children were divided into group A (bloodstream infection group) and group B (pulmonary infection group) according to the infection foci, and the efficacy of mNGS and traditional pathogen detection (TPD) in the two groups was compared. Results A total of 56 children (27 boys and 29 girls) were included, with a median age of 4.0 (2.0-7.8) years. There were 39 cases in group A (28 cases of ALL and 11 cases of AML) and 17 cases in group B (10 cases of ALL and 7 cases of AML). The commonest pathogens in group A and B were Aspergillus, Candida and Acinetobacter baumannii. The consistency of positive rate of mNGS and TPD was poor (Kappa=0.039). The mNGS positive rate (80.4%) was significantly higher than TPD (58.9%), and the difference was statistically significant (P=0.023). In group A, the positive rates of mNGS for Aspergillus (35.9% vs. 12.8%), Candida (28.2% vs. 10.3%) and Acinetobacter baumannii (28.2% vs. 7.7%) were higher than those of TPD; in group B, the positive rate of mNGS for Aspergillus (41.2% vs. 5.9%) was higher than that of TPD, and the differences were statistically significant (P<0.05). The mNGS can detect rare pathogens, and the detection rate of mNGS for mixed infection was significantly higher than that of TPD, and the difference was statistically significant (P<0.05). Conclusions In leukemia children with FN who failed in initial antibiotic therapy, the commonest pathogens were Aspergillus, Candida, and Acinetobacter baumannii. As a complementary tool for TPD, mNGS has a higher positive detection rate for common pathogens and outstanding efficacy for mixed infections and rare pathogens.