Alcohol is an underestimated toxicant, and binge drinking is increasing rapidly. Macrophages are important immune cells characterized by heterogeneity and can be polarized into pro-inflammatory and anti-inflammatory phenotypes.To elucidate the effects of acute alcohol exposure on macrophage polarization and function, we established an optimized multi-parameter flow cytometry panel, assessed the impact of acute alcohol on macrophage polarization, developed macrophage polarization models, and proposed novel indices for a refined understanding of macrophage polarization status.First, we developed a flow cytometry panel quantifying eleven markers critical to macrophage polarization within the RAW 264.7 cell line. Inducing specific polarization states with IFN-γ ± LPS for M1 macrophages and IL4 and IL10 for M2a and M2c macrophages ensured a consistent environment for studying macrophage activation and polarization. Second, we assessed the in vitro effects of 86.8 mM alcohol treatment on RAW 264.7 cells, approximating severe human alcohol consumption. The results indicated that acute alcohol exposure compromises macrophage functionality across M1 and M2 roles by increasing toxicity, reducing viability, and impairing innate immune recognition. Third, we studied the in vivo effects of acute alcohol treatment on peritoneal macrophage polarization in BALB/c mice. Administering 6 g/kg of alcohol resulted in a peak blood ethanol concentration around 86.8 mM. Acute alcohol had diminished suppressive effects on LPM and SPM polarization compared to in vitro study, partially suppressing TLR4, CD14, CD86, Arginase-1, and VEGF in LPMs, and altering TLR4, MHC II, CD86, and Arginase-1 in SPMs. Finally, using PCA and UMAP, we identified key markers like CD86, CD206, MHC II, TNF-α, and IL10, introducing the CD86/CD206 ratio and the LPM/SPM index as novel indices to measure macrophage polarization. This study provides insights into how acute alcohol exposure affects the innate immune system, particularly macrophage polarization and function. These findings lay the groundwork for future research to mitigate the adverse effects of alcohol on the immune system, offering valuable tools for studying macrophage biology and the impact of external factors on immune function.