This study aims to design and develop a set of universal primers for amplification of the entire mitochondrial genome of Acaridae mites based on polymerase chain reaction (PCR). The conserved regions were screened by comparing the mitochondrial genomes of known Acaridae mites in GenBank. To ensure that the primers cover the entire mitochondrial sequence, the mitochondrial genome was divided into six overlapping fragments based on the conserved region. After synthesizing primers, we extracted the total DNA of Acaridae mites collected from grain stores. Then the total DNA of individual Acaridae mites was used as the template for PCR, Sanger sequencing, and sequence splicing. Agarose gel electrophoresis revealed that the lengths of the six amplification products were between 1.2 kb and 3.8 kb, consistent with design expectations. The resulting sequences could be successfully spliced to form a loop structure, confirming that the primers cover the entire mitochondrial genome. The splicing results were consistent with the full sequence released in GenBank. Developed universal primers can achieve rapid amplification and help to obtain mitochondrial genome from a single acarid mite with high efficiency.