The Joule heating effect on electroluminescent efficiency is important in the degradation origin of organic light-emitting diodes (OLED). Scanning tunneling microscopy (STM) and photoluminescence (PL) measurements were performed on the guest molecule BT (1,4-bis(benzothiazole-vinyl) benzene), host molecule TPBI (2, 2',2"-(1,3,5-phenylene)tris-[1-phenyl-1H-benzimidazole]), and their mixture deposited on an HOPG surface to study the OLED degradation mechanism due to thermal heating. At room temperature, BT and TPBI in the mixed layer show good compatibility and high PL intensity, but at higher temperatures, they show phase separation and aggregation into their own domains and a concomitant decrease in PL intensity. The PL intensity loss suggests ineffective energy transfer from TPBI to BT due to phase separation, which may cause OLED degradation. Scanning tunneling spectroscopy (STS) results show that the band gaps of TPBI and BT remain unchanged with the annealing temperature, suggesting that the heat-induced decay of OLED is related to the interfacial structural change rather than the respective molecular band gap. The results provide direct evidence showing how the molecular structures of the mixed layer vary and affect the PL intensity due to temperature.