Rapid, simple, accurate and highly sensitive detection of enzymes is essential for early screening and clinical diagnosis of many diseases. In this study, we report the fabrication of a turn-on ratiometric electrochemical sensor for the in situ determination of β-Galactosidase (β-Gal) based on surface engineering and the design of a molecular probe (Pygal) specific for β-Gal recognition. First, Pygal probe was synthesized and characterized, and then co-assembled with the methylene blue (MB) internal reference probe on the surface of single-wall carbon nanotubes (SWCNT)-modified carbon fiber microelectrode (CFME). The resulting CFME/SWCNT/MB + Pygal sensor is activated in the presence of β-Gal giving one peak at 0.33 V originating from the oxidation of the product of Pygal enzymatic hydrolysis (PyOH). Another oxidation peak attributed to MB appears simultaneously at −0.28 V allowing the construction of a ratiometric electrochemical sensor for β-Gal detection with improved sensitivity and accuracy. The sensor showed a linear response to β-Gal in a wide concentration range from 1.5 to 30 U L−1 and a low detection limit of 0.1 U L−1. Moreover, the sensor demonstrated excellent selectivity against several biologically relevant hydrolases and redox-active molecules. Finally, the combination of excellent electrochemical performance and favorable physicochemical properties of CFME allowed the determination of β-Gal in the whole blood of Parkinson's Disease (PD) model mice. The workflow reported in this study provides a strategy for the design and development of sensors for the in vivo monitoring of other enzymes important for the early diagnosis of different health issues.