The interaction mechanism of amlodipine (AD) and lactate dehydrogenase (LDH) was examined using different spectroscopic and molecular docking methods. Spectroscopy results demonstrated that AD could bind with LDH, thus inhibiting its activity in a time-dependent and dose-dependent manner. Based on the thermodynamic parameters, it was concluded that the reaction between LDH and AD occurred spontaneously, where H-bonding and van der Waals force occupied vital roles. The association constant (K) was the order of magnitude of 105, the number of binding site (n) was approximately 1, and binding distance between AD and LDH was estimated as 3.5 nm. Synchronous fluorescence, circular dichroism, and FT-IR spectra indicated that AD led to conformational variations in LDH. The results from molecular docking found that AD was inserted into the hydrophobic cavity of LDH and interacted with residue His192, resulting in the inhibition of LDH activity. The results of Discovery Studio Visualizer highlighted the role of H-bonding in the binding process, in agreement with the solution experiments. This work would help in understanding the binding mode of LDH-AD system and would provide new insights into the effect of AD on human health.