The invention discloses a sequencing library construction method and kit for pathogenic microorganism detection. The method comprises: (1) putting an extracted sample into a sample preservation solution, carrying out DNA / RNA co-extraction, and removing host rRNA; (2) adding reverse transcriptase to enable RNA in the sample to be subjected to reverse transcription to form RNA / cDNA composite doublestrands; (3) directly adding mutant Tn5 transposase and a breaking buffer solution into a mixture of the reverse transcription product cDNA / RNA composite double strand and a DNA double strand for fragmentation, and after the fragmentation is finished, carrying out a termination reaction by using a termination reaction solution 5*TS-plus; (3) adding an amplification buffer solution and a strand displacement and amplification enzyme mixture, and carrying out PCR amplification; and (4) carrying out magnetic bead purification to obtain a sequencing library of the original DNA / RNA. According to themethod, library building steps are greatly reduced, the library building time is shortened, the requirement on the initial quantity of samples is reduced, and the library output and offline data quality are improved.