The invention discloses an in-vitro diagnostic check test strip and a manufacturing method thereof. The in-vitro diagnostic check test strip comprises a substrate, a reaction membrane and a mat, wherein, the substrate is adhered with the reaction membrane and the mat, one end of the reaction membrane is mutually connected with the mat in a lapping joint way, the substrate is made of transparent plastic or PVC material, the surface of the substrate, which is adhered with the reaction membrane, is provided with parallel checking line and quality-control line, and the mat is coated with colloidal gold solution or colored latex solution with the diameter range of 10nm to 50nm; the manufacturing method comprises the steps of preparation of the reaction membrane, marking and solid phase of the mat, assembly and cutting. The invention has the advantages of being capable of judging the testing result directly by the transparent substrate, preventing the volatilization of liquid and the infiltration of takeout liquid, causing the testing result to be more accurate, reducing nonspecific adsorption during testing reaction, reducing reaction background, preventing tracer from being adsorbed on the reaction membrane, guaranteeing fast elution and release, and being simple and convenient and feasible.