We aim to develop an in situ microfluidic biosensor based on laccase from Trametes pubescens with flow-injection and amperometry as the transducer method. The enzyme was directly immobilized by potential step chronoamperometry, and the immobilization was studied using cyclic voltammetry and electrochemical impedance spectroscopy. The electrode response by amperometry was probed using ABTS and syringaldazine. A shift of interfacial electron transfer resistance and the electron transfer rate constant from 18.1 kΩ to 3.9 MΩ and 4.6 × 10 −2 cm s −1 to 2.1 × 10 −4 cm s −1 , respectively, evidenced that laccase was immobilized on the electrode by the proposed method. We established the optimum operating conditions of temperature (55°C), pH (4.5), injection flow rate (200 µ L min −1 ), and applied potential (0.4 V). Finally, the microfluidic biosensor showed better lower limit of detection (0.149 µ M) and sensitivity (0.2341 nA µ M −1 ) for ABTS than previous laccase-based biosensors and the in situ operation capacity.