Cluster of differentiation 147 (CD147), a transmembrane glycoprotein, has been identified as a potential auxiliary receptor for the SARS-CoV-2 spike protein (SP), contributing to COVID-19 infection. However, the detailed binding characteristics of this interaction remain unclear. Here, we characterized SP-CD147 binding using Enzyme-Linked Immunosorbent Assay (ELISA) and single-molecule force spectroscopy (SMFS) under varying contact times and temperatures. While SP binds CD147 with lower force than angiotensin-converting enzyme 2 (ACE2) at normal body temperatures, its binding strength to CD147 significantly increases under fever-like conditions, contrasting with SP-ACE2 interactions, which weaken at elevated temperatures. We further investigated the role of heparin in modulating this interaction and found that heparin independently binds both CD147 and SP, enhancing the interaction force between CD147 and SP, as confirmed by Quartz Crystal Microbalance (QCM) and SMFS. Molecular dynamics simulations and binding free energy calculations provided atomic-level insights into the stabilizing role of heparin within the SP/CD147 complex. These findings offer a comprehensive characterization of the CD147-SP interaction, revealing potential therapeutic avenues for COVID-19 intervention.