Lactic acid is a by-product of glycolysis prevalent in the body fluid and significant in a living cell, it is also an important biomolecule that exists in human sweat. Lactic acid is firmly interlinked with various diseases for the direct detection by the technique of current is challenging. Hence, it is important to develop an enzyme-free electrochemical sensor for it. A monometallic salt magnesium-(3-hydroxypyridine-2-carboxilic acid) metal-organic framework anchored at carbon nanofiber mat in the solvothermal process. As prepared Mg(HPCA)MOF@CNF has a good surface area within high porosity and also possesses hydrophilicity, facilitating interaction between the analyte molecule and the active metal site of the MOF network for the redox process, which facilitates fast electron transfer with low resistance, which results in excellent sensitivity. The catalytic activity of MOF was improved in a basic medium, i.e., in pH 8 buffer solution, with excellent electrochemical sensitivity towards lactic acid. The redox reaction was diffusion-controlled and irreversible between lactic acid and hybrid mat. Mg(HPCA)MOF@CNF/GCE depicted the linear range of 0.1–5 mM, and the result of the lower limit of detection is 2.5 μM. The hybrid mat has good interference properties of some species such as sodium hydroxide, potassium chloride, hypoxanthine, xanthine, uric acid, dopamine, and citric acid, bending ability, and good storage capability for up to 35 days. Mg(HPCA)MOF@CNF/GCE demonstrates a significant sensor for the detection of lactic acid in human sweat under natural environments with accuracy and reliability. This work can lead to manufacting innovation in the future.