Cell-free DNA is naturally degraded in various bodily fluids. The aim of this study was to determine the degradation kinetics of DNA, with and without protein, in serum, urine and saliva. Naked DNA and DNA-protein complex were prepared, added to the samples to be analysed and incubated at 37°C and room temperature for various lengths of time. Alleles of 20 short tandem repeat loci were amplified from the incubated samples, and clearance models were generated from the mean peak areas. Plotting the natural logarithm of DNA concentration against the incubation time produced a linear relationship. The half-lives of DNA with and without protein in serum were 157.6min and 30.8min at 37°C, 330.5min and 70.5min at room temperature, respectively. The half-lives of DNA with protein in saliva were 175.6min and 251.3min at 37°C and room temperature, respectively. However, the half-lives of DNA in urine (both with and without protein) were too short to detect. The kinetics of DNA degradation in serum and saliva followed a first-order clearance model. Urine had the strongest effect on DNA degradation, and the half-lives of DNA with protein were relatively longer than those of naked DNA. •DNA degradations were qualitatively observed in serum, saliva and urine.•DNA degradation followed a first-order clearance model in these samples.•The half-lives of DNA with protein were relatively longer than those of naked DNA.•The half-lives were around 3h in 37°C and 5h in 24°C for serum and saliva.•The half-lives of DNA in urine were too short to detect in room temperature.