The continuous real-time monitoring of human health using biomedical sensing devices has recently become a promising approach to the realization of distant health monitoring. In this paper, the piezoelectric characteristics of the silk fibroin (SF) natural polymer were analyzed as the material used for obtaining sensing information in the application of distance health monitoring. To enhance the SF piezoelectricity, this paper presents the development of a novel SF-based sensor realized by combining SF with different carbon nanofiber (CNF) densities, and for such newly developed SF-based sensors comprehensive performance analyses have been performed. Versatile methods including the scanning electron microscope, Fourier transform infrared spectroscopy, Raman and X-ray diffraction measurements and impedance analysis were used to study the morphologic, mechanical and electrical properties of the developed SF-based sensor. The SF with CNF samples was analyzed for three different pressure loads (40 N, 60 N and 80 N) in 500 compression test cycles. The analyses thoroughly describe how combining natural polymer SF with different CNF densities impacts the piezoelectricity and mechanical strength of the proposed SF-based sensor. The developed piezoelectric SF-based sensors were further tested on humans in real medical applications to detect generated piezoelectric voltage in versatile body movements. The maximum piezoelectricity equal to 2.95 ± 0.03 V was achieved for the jumping movement, and the SF sample with a CNF density equal to 0.4% was tested. Obtained results also show that the proposed SF-based sensor has an appropriate piezoelectric sensitivity for each of the analyzed body movement types, and that the proposed SF-based sensor can be applied in real medical applications as a biomedical sensing device. The proposed SF-based sensor’s practical implementation is further confirmed by the results of cytotoxicity analyses, which show that the developed sensor has a non-toxic and biocompatible nature and can be efficiently used in skin contact for biomedical wearable health monitoring applications.