The invention discloses a method and device for detecting instability of a microsatellite locus, wherein the method comprises the steps of S1, treating a sample; S2, processing data, to be specific, using comparison software to compare determined sequences to a reference genome to obtain a comparison file, allowing non-matched sequences to form soft cut-off, ranking according to comparison positions, and using the software samtools to establish an index; S3, detecting the instability of the microsatellite locus, to be specific, using locus coverage rates having significant differences to determine the microsatellite instability of the sample. By using the method and device in connection with high-throughput sequencing techniques for capturing a target area, the quantity of loci for detection can be increased greatly; differences between a pathological sample and a control sample are judged, the microsatellite stability of the sample is determined through the differences in connection with statistical testing, and chances for false positives and false negatives to occur in the detection of microsatellite locus stability in the prior art are slimmed.