Stored-food and house-dust arthropods include many species of mites and beetles that affect human health. For diagnostic tests proteases such as trypsin are utilized as they are indicators of the presence of allergen contaminants in food. We recently characterized Kunitz-type protease inhibitors (KPIs) from Solanum palustre. Here we studied biotechnological applications of KPI-B1 and -B4. We manufactured a protein chip with immobilized KPI-B1 and -B4 and showed trypsin/chymotrypsin-binding specificity, indicating that the recombinant proteins have protease selectivity. We employed the protein chip to capture mite proteins belonging to the protease family with polyclonal anti-mite antibodies. The mite diagnostic chip can be useful for detecting mite allergens.