The invention relates to an on-site collection method of a karst cave water environment DNA sample, which comprises the following steps: 1, collecting water from a karst cave water environment DNA detection point by using a Bailer tube, and putting the collected water sample into a disposable plastic basin for later use; 2, preliminarily filtering the DNA water sample in the karst cave environment collected in the step 1, and removing large-particle impurities, 3, taking a disposable syringe, sucking the preliminarily filtered water sample, connecting the water sample to a membrane-replaceable needle type filter loaded with a glass fiber membrane, manually extruding the syringe at a constant speed to enable the water sample to pass through the membrane-replaceable needle type filter, and then discharging the water sample, putting the replaced filter membrane into a centrifugal tube filled with anhydrous alcohol, and storing at low temperature. According to the method, the DNA sample in the karst cave water environment can be collected on site, the problems of DNA degradation, cross contamination and the like when a water sample is remotely transported to a laboratory for vacuum filtration are solved, and the collection method is low in cost, simple and convenient to operate and high in accuracy of the collected sample.