Objective To compare the quality between the glass specimens of acaroid mites prepared with three mounting media. Methods Polyethylene lactic acid phenol medium, Berlese medium, and Hoyer's medium were prepared according to formulae, and their physical and chemical properties were compared. Sixty glass specimens of acaroid mites were prepared with each medium and were observed under a optical microscope after being oven-dried. The obtained data were analyzed by chi-square test. Results Polyethylene lactic acid phenol medium was light yellow liquid, with a density of 0.99 g/ml. Berlese medium was reddish brown, with a density of 1.12 g/ml. Hoyer's medium was slightly lighter than Berlese medium, with a density of 1.37 g/ml. The optical microscopy showed that the glass specimens of acaroid mites prepared with Hoyer's medium had high transparency and stretched mite bodies, with little yellowing and blackening at both sides of mite bodies; the specimens prepared with the other two media had relatively low quality. The chi-square test revealed significant differences between the three media (χ²=15.54, P<0.01), and the specimens prepared with Hoyer's medium had significantly higher quality than that prepared with polyethylene lactic acid phenol medium (χ²=7.50, P<0.01) and that prepared with Berlese medium (χ²=15.70, P<0.01). Conclusion Glass specimens of acaroid mites prepared with Hoyer's medium have the best quality under a optical microscope, providing a reference for the preparation of high-quality glass specimens of acaroid mites.