The invention discloses a test kit of HSP70 double antibody sandwich method used in the sample drawn from human, big mice and small mice, and comprises a kit body, an enzyme-labeled plate of solidified HSP70 monoclonal antibody provided in the kit body and substances provided in the kit body; the substances provided in the kit body comprises a HIS-HSP70 protein standard reagent, a HSP70 polyclonal antibody labeled by biotin, a sample dilution solution, a cleaning buffer solution, an antibody solution, a horseradish enzyme-labeled avidin, a horseradish enzyme-labeled avidin dilution solution, a substrate visualization solution A solution, a substrate visualization solution B solution and a stop solution. The invention has the advantages of high detection sensitivity, big accuracy and low cost; the invention can detect the HSP70 protein in a cell lysis solution, a tissue extracting solution, plasma and serum drawn from human, big mice and small mice at the same time.