This work presents a new design of miniaturized microneedle pH sensors based on AgIO3/Ag/polytetrafluoroethylene (PTFE)/WO3/tungsten (W), which can be used to measure the real-time label-free pH of various micro-sized cells and small-volume bodily fluids. The fabrication process comprises multiple steps, including creating W microneedles and depositing AgIO3/Ag/PTFE/WO3 layers. A needle-like structure was created at the end of a cleaved W rod using electrolytic polishing. The sensing layer (working electrode, WE) of WO3 films was developed onto W microneedles using the thermal oxidation method. A PTFE coating was applied as an insulating layer between the WE and the reference electrode (RE, AgIO3/Ag thin film). The PTFE and AgIO3/Ag layers were fabricated using the hand spray and RF sputtering methods, respectively. The microstructural and compositional features of miniaturized microneedle sensors (AgIO3/Ag/PTFE/WO3/W) were analyzed using field emission scanning electron microscopy (FESEM), energy dispersive X-ray (EDX), X-ray diffractometer (XRD), and Raman techniques. The miniaturized microneedle sensors exhibited excellent analytical performance, including a Nernstian slope of −49.36 mV/pH and a linear response range from pH 4.01 to pH 9.18, as verified by testing with a pH standard solution. Ex-vivo pH testing results of fish eggs demonstrated the ability of the fabricated miniature microneedle pH sensor to penetrate biological tissue and accurately measure pH values.