A fast mixed-signal readout circuit for high sensitivity organic vertical nano-junctions (VNJ) ammonia sensor detection is proposed in this study. Accompanying with the ammonia sensor, a low power, fast response and high resolution readout circuit is designed and successfully taped-out in this study. The analog front-end circuit includes a pre-amplifier, a sample and hold (S/H) circuit, a differential current, a pulse width modulation (PWM) circuit, an automatically sensing and reset (S/R) circuit. The digital back-end is realized by cell-based technology to compute and display concentration values instantaneously. The chip is fabricated by the TSMC 0.18-μm 1P6M 3.3V mixed-signal CMOS process technique for verification. The chip area is 1.5 × 1.6 mm2 while power consumption is 3.2 mW. Experiment results show a strong linear relationship exists between differential S/R output slope and the gas concentration level in a logarithm scale, which is critically beneficial to sensor design. In results, the readout circuit has fast responses, 50 seconds, based on the auto-sensing circuit catching different slope coefficient of the signal in transient time. The resolution is 70.48mV/log(ppm) while error rate, average noise and detection rate reliability are 2.86%, 123 μVrms, and 99.6%, respectively. This chip could be suitable for applications in cars, cell phones, watches, etc.