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This paper presents a CMOS ion-sensitive field-effect transistor (ISFET) sensor for Escherichia coli (E. coli) level detection. Non-ideal effects, such as passivation attenuation, trapped charge, and parasitic capacitance, lead to sensor non-uniformity issue. To alleviate it, a subthreshold pH-to-Time-to-Voltage Conversion (pH-TVC) readout structure with digital double sampling (DDS) and pixel level calibration are employed. The measured gain after calibration was 118.7 mV/pH with 0.01 pH resolution. In addition, three levels of E. coli samples were prepared and cultured for screening. Experimental result showed that E. coli levels can be differentiated within 4 hours. It had better time efficiency compared to conventional direct plate counting method (24-h). As such, the proposed high-sensitivity ISFET sensor has great potential in the portable, accelerated and low-cost food safety screening.