Incorporating silk fibroin (SF) into the biosensing platform provides a level of flexibility and macro-structure control that holds immense importance for monitoring of sweat pH. It accomplishes non-invasive surveillance of the human physiology while concurrently tackling the issue of electrode-skin friction. The preparation of the multi-walled carbon nanotubes (MWCNTs)/polyaniline (PANI) silk fibroin pH-responsive microneedle array sensor involves the dispersion of PANI powder in silk fibroin and the addition of MWCNTs for the analysis of human sweat. The template method offers an efficient and straightforward means of producing microneedles with a high degree of precision, achieving micrometer resolution. The pH sensor is designed with a two-electrode configuration, comprising an MWCNTs/PANI silk fibroin microneedle array sensing electrode as well as an Ag/AgCl reference electrode. The pH sensor shows a sensitivity of 53.60 mV/pH over the pH range 3 to 9, a response time of 68 s, and a recovery time of 77 s. The pH sensor displays other outstanding sensor performances regarding reversibility, repeatability, selectivity, and stability. The pH sensor is capable of detecting alterations in the pH levels of human sweat.