The invention relates to the field of microorganism and molecular biology, and in particular relates to a method for detecting trace fungus by using single-cell sequencing and a fungus detection kit produced by using the method. The method comprises the steps of extraction of trace fungus cells, extraction of fungus protoplasm by fragmentation of fungus cell wall, extraction and amplification of trace fungus protoplasm gDNA, database creation of gDNA, genome sequencing, bioinformatic analysis comparison, and judgment on the type of the detected fungus. The method disclosed by the invention realizes effective detection on trace fungus, and can be directly applied to separation, detection and identification of trace difficult fungus samples or mixed samples and deep study of genetic information. The fungus detection method and the kit are applicable to the fields of industrial production, environmental monitoring, air detection, soil detection, water quality detection, food detection, drug detection, cosmetic detection, health care products detection and medical detection.