The detection of pH/temperature at the single-cell level is hoping for the great level of clinical importance for the early detection of many diseases like cancer, diabetes, etc. In this research work, we have fabricated a micro region pH/temperature sensors by series of processes like electrolytic polishing to create needle structure, deposition of electrode materials using RF magnetron sputtering for pH measurements and finally testing in various biological mediums. Working and reference electrodes were Ag/AgIO3 and Sb/Sb2O3 deposited on microneedles under optimized deposition parameters. The structural, elemental and morphological properties were analyzed using XRD, XPS, EDS and FE-SEM. The fabricated tip of the microneedle probe is around analyzed by FE-SEM which size is comparable with the biological cells. pH testing was initially begin with using fish egg, various biological cells and further in vivo tested in mice cerebrospinal fluid and bladder. The obtained temperature/pH sensing results were adequate with theoretical values. Since the sensor works at micro region, the potential difference is easily disturbed by atmospheric anomalies. Hence, many steps have been taken to improve the stability of the sensor.