The present invention provides a method for microbial detection based on exosome nucleic acid and application thereof. The method comprises: (a) separating and obtaining exosome nucleic acid from a sample to be tested, the sample to be tested coming from first species; (b) sequencing the exosome nucleic acid to obtain a sequencing result; (c) excluding nucleic acid sequences corresponding to the first species from the sequencing result, so as to obtain sequence data subjected to removal treatment; and (d) comparing the sequence data subjected to removal treatment with nucleic acid sequences ina microbial database to obtain a microbial detection result in the sample.