A biosensor according to the present invention comprises a support 1, a conductive layer 2 composed of an electrical conductive material such as noble metal, for example gold, palladium or the like, and carbon, slits 3a and 3b parallel to the side of the support 1, slits 4a and 4b vertical to the side of the support 1, a working electrode 5, a counter electrode 6, a detecting electrode 7, a spacer 8 which covers the working electrode 5, the counter electrode 6 and the detecting electrode 7 on the support 1, a rectangular cutout part 9 forming a specimen supply path, an inlet 9a of the specimen supply path, a reagent layer 12 formed by applying a reagent including an enzyme or the like to the working electrode 5, the counter electrode 6 and the detecting electrode 7, which is exposed from the cutout part 9 of the spacer 8, and a cover 13 which covers the spacer 8, as shown in FIG. 1. The so-constructed biosensor can be formed by a simple method, and a biosensor which is excellent in a measuring accuracy as well as a biosensor in which a reagent layer is placed uniformly on the electrodes regardless of a reagent liquid composition, and which has a uniform performance can be realized.