The present invention provides a power source-free and valve-free type single molecule detection chip, which comprises a reaction layer, a modification layer, a sealing layer, a main channel, small reaction chambers, a sample inlet, a sample outlet and a heat resistant adhesive tape, wherein the reaction layer is formed by connecting the main channel and the reaction small chambers in series, the reaction small chambers are positioned above or below the main channel, the sample inlet and the sample outlet are positioned on both ends of the main channel, the modification layer is pre-connected with the sealing layer, the reaction layer is sealed with the sealing layer through the modification layer to form a microfluidic chip assembly, and the heat resistant adhesive tape is adhered on the upper surface of the reaction layer. According to the present invention, miniaturization is achieved, such that consumption of reagents and samples are reduced, and cost is low; a small amount of liquid can be assigned to hundreds of the independent small chambers without an external connection drive system and valve switching control equipment so as to reduce complexity of chip production and use; and the operation is simple, other control devices are not required, application cost is low, and the chip can be used in real-time fluorescence quantitative polymerase chain reactions and digital nucleic acid amplification.