The temperature effect has been discussed in the past on pH sensors. In this study, the temperature effect is analyzed for the ascorbic acid (AA) biosensor based on enzymatic ascorbate oxidase (AO). To reduce this effect, a novel temperature compensation circuit was implemented by Taiwan Semiconductor Manufacturing Co., Ltd. (TSMC) 180 nm complementary metal-oxide-semiconductor (CMOS) process. By applying the proposed circuit, the temperature coefficients (TCs) with five concentrations of AA were suppressed below 201 μV/°C. The TC of the normal level of AA concentration (0.0312 mM) in the human body was 188 μV/°C, at the temperature range from 25°C to 55°C. The polyethylene terephthalate (PET) substrate was utilized. To confirm the stability of the fabricated AO/RuO2/PET AA biosensor, interference and hysteresis experiments were carried out. At present, the experiments were still in the stage of in vitro measurement. The results showed that the AO/RuO2/PET AA biosensor had good selectivity that effective against other interference that may exist in blood at either 25 °C or 37.5 °C. Moreover, at the temperature of 37.5 °C, the hysteresis voltage of the biosensor after applying the compensation circuit was reduced from 18.27 mV to 1.39 mV.