Electrochemical methods have been shown to be advantageous to life sciences by supporting studies and discoveries in metabolism activities, DNA analysis, and neurotransmitter signaling. Meanwhile, the integration of Microelectrode Array (MEA) and the accessibility of CMOS technology permit high-density electrochemical sensing method. This paper describes an electrochemical imaging system equipped with a custom CMOS microchip. The microchip holds a 3.6 mm × 3.6 mm sensing area containing 16,064 Pt MEA, the associated 16,064 integrated read channels, and digital control circuits. The novel three-electrode system geometry with a 27.5 μm spatial pitch enables cellular level chemical gradient imaging of bio-samples. The noise level of the on-chip read channel array allows amperometric detection of neurotransmitters such as norepinephrine (NE) with concentrations from 4 μM to 512 μM with 4.7 pA/μM sensitivity (R2 = 0.98). Electrochemical response to dissolved oxygen (DO) concentration was also characterized by deoxygenated deionized water containing 5% to 80% of the ambient oxygen concentrations with 86 pA/mg/L sensitivity (R2 = 0.89). The system also demonstrated selectivity to different target analytes using cyclic voltammetry method to simultaneously detect NE and uric acid. Also, a custom indium tin oxide with deposited Au glass electrode was integrated into the microfluidic system to enable pH measurement, ensuring the viability of bio-samples during experiments. Electrochemical images confirm the spatiotemporal performance at four frames per second while maintaining the sensitivity to target analytes. Finally, the overall system is controlled and continuously monitored by a MATLAB-based custom user interface, which is optimized for real-time high spatiotemporal resolution chemical imaging.