The precise manipulation of chiral crystallization for the fabrication of homochiral surfaces has emerged as a pivotal focal point within the realm of surface chemistry. In this investigation, we employed a temperature-regulated strategy, utilizing the conformationally chiral molecule 1,1,2,2-tetrakis(4-bromophenyl)ethene (TPEB) as a precursor to control the chiral recognition and separation process on Ag(111) surface mediated through Br···H and Br···Br interactions. At a temperature of 77 K, when TPEB was deposited onto the Ag(111) surface, it gives rise to two mirror-image racemic structures. As the temperature increases, TPEB molecules undergo a distinct transition from existing in racemic mixtures to enantiomeric excess mixtures. This transition ultimately culminates in a chiral separation process, resulting in the formation of a single-handed self-assembled structure and the acquisition of a homochiral Kagomé lattice. This work meticulously monitored the chiral crystallization process of the conformationally chiral molecules on Ag(111) surface, capturing structures with diverse enantiomeric compositions. Moreover, through a combination of molecular simulations and density functional theory (DFT) calculations, we elucidated that an increase in temperature enhances the recognition between chiral molecules of the same handedness. This enhanced recognition, in turn, promotes the chiral separation process and steers the transition of the supramolecular assemblies from racemic mixtures towards single-handed structures. This study not only achieved the separation of heterochiral molecular conformations but also paved an effective avenue for the fabrication of homochiral nanostructures.