The invention provides an optical biosensor used for detecting surface cleanliness rapidly, and a testing method thereof. The sensor comprises a sampling stick, a detection cavity, a liquid storage tank and a reaction chamber. The sampling stick comprises a handle, a spacing ring, a sampling swab and a pointed casing; the liquid storage tank is provided with a plurality of uniform sealing pieces which are in fixed connection through screw threads, and an ATP extractant and a neutralization reagent used for neutralizing the remaining extractant are sealed therein; and the lateral wall of the reaction chamber allows light to pass through, and a sensitive reagent is fixed at the bottom part in advance. When in detection, firstly, the sampling swab of the sampling stick is used for daubing and sampling the surface of an object to be detected; then, the pointed casing is screwed downwards; the sampling stick is inserted into the sampling detection cavity; and with the consideration of the fluorescence strength during the detection of a sample by an optical device, the surface cleanliness of the object to be tested can be determined by comparing the light intensity value with the standard sanitary threshold value. The sensor provided by the invention has the advantages of simple operation, convenient use, rapid response and disposability, and can be widely applied to the object surface cleanliness real-time detection and the field hygiene supervision in industries, such as food processing, cosmetics production, medical service and the like.