The purpose of this study was to evaluate the effect of temperature and time of sous-vide cooking method on the characteristics of Thoroughbred horse loin. Sliced portions (200 ± 50 g) were cooked by boiling (control) and sous-vide (65 and 70 °C for 12, 18, and 24 h). The samples were analyzed for proximate composition, pH, color, texture, microstructure, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), microbiology, volatile organic compounds (VOCs), nucleotide content, and fatty acids composition. The color analysis showed decreased redness at elevated temperatures. Improved tenderness, demonstrated by reduced shear force values (36.36 N at 65 °C for 24 h and 35.70 N at 70 °C for 24 h). The micrographs indicated dense fiber arrangements at 70 °C. The SDS-PAGE revealed muscle protein degradation with extended sous-vide cooking. The VOC analysis identified specific compounds, potentially distinctive markers for sous-vide cooking of horse meat including 1-octen-3-ol, decanal, n-caproic acid vinyl ester, cyclotetrasiloxane, octamethyl, and 3,3-dimethyl-1,2-epoxybutane. This study highlights the cooking time’s primary role in sous vide-cooked horse meat tenderness and proposes specific VOCs as potential markers. Further research should explore the exclusivity of these VOCs to sous-vide cooking.