Multiple sensor electrodes, a supplementary electrode, a reference electrode, and signal-processing circuits were integrated on a single chip to develop a chip-shaped electrochemical sensing system. L-lactate and glucose were measured using on-chip working electrodes modified by polyion complex to immobilize lactate oxidase and glucose oxidase, respectively. Cyclic voltammetry measurements were conducted using an on-chip potentiostat. Selective and quantitative detection of glucose and L-lactate and the interference behavior were studied. Hydrogen peroxide generated by enzymatic reactions was detected by an increase in anodic oxidation current. Reaction currents at +0.7 V versus Ag/AgCl were used to obtain calibration plots. The measured dynamic ranges for L-lactate and glucose were 0.2–1.0 mM and 2.0–8.0 mM, respectively. The sensitivities were 65 nA/mM and 15 nA/mM, respectively, using a working electrode of 0.5 mm2. The 3σdetection limit was 0.19 mM and 1.1 mM, respectively. We have achieved multiple biomaterial detections on a circuit-equipped single chip. This integrated electrochemical sensor chip could be the best candidate for realizing point-of-care testing due to its portability and potential for mass production.