In this study, we present the fabrication and characterization of a microfluidic pH sensing chip designed to detect human lung cancer (A549) cells. The chip utilizes a Sb2O3/Sb thin film as the working electrode and an AgIO3/Ag thin film as the reference electrode, both deposited using the radio frequency (RF) sputtering technique. The PDMS microfluidic channel was prepared using a 3D-printed mold, followed by curing of PDMS within the mold. The fabricated sensing chip demonstrated excellent linear response (R2 ∼ 0.99) and appreciable Nernstian sensitivity (average = -57.02 ± 1.23 mV/pH, n = 4) in a wide pH range of 1.68–10.01. It exhibited good stability over a 48-hour duration with acceptable drift voltage and negligible temperature impact on the sensing performance. Further, in-vitro pH testing of human lung cancer (A549) cells was performed to investigate the real-time pH measurement ability of the developed microfluidic pH sensing chip.