Human Lactate Dehydrogenase (LDH) is an oxidoreductase enzyme that catalyzes the conversion of lactate to pyruvate under anaerobic conditions. The LDH test, a marker for acute or chronic diseases, uses the human LDH amino acid sequence to identify tissue injury sites in bodily tissues. The evolutionary relationship of human LDH to other species was investigated. Manifold molecular modeling was used to choose the best-modeled 3D LDH structure. Among the 4 chains, each chain has a set of parallel running β-sheets interspersed with helices and coils. Ramachandran plot was used to validate the stereochemical parameters. To improve the stability of the protein, loop optimization was followed by energy minimization. Thermodynamic spontaneity (ΔG value) and net area for solvent accessibility showed the post-minimized structure to be the most stable one. The optimization process resulted in a decrease in the percentage of coils, indicating a steady conformation with flexibility to interact with partner proteins. The domain area was also studied to have binding pockets. The residues in binding pockets were highly conserved throughout the evolution and formed mainly sheet conformation. The study opens new research opportunities for human LDH drug discovery, revealing numerous interaction partners and necessitating further molecular-level study.