The solid β-cyclodextrin (β-CD) was selected as the separating medium of chromatographic column to separate the isomers, 3-biphenylcarboxylic (3-BPCA) and 4-biphenylcarboxylic (4-BPCA). Separation capability for different solvents (ethanol, ethyl acetate, chloroform, toluene, and hexane) was investigated, and was assessed based on the separation factor (S). The results showed that hexane was considered as the best solvent due to the high separation factor of 1.525 and a sharper eluent peak compared to the other solvents. Ethanol could dissociate 3-BPCA and 4-BPCA from β-CD completely due to its strong polarity, so ethanol was selected as the reverse solvent. This study provided satisfactory results on the purity and yield for the separation of 3-BPCA and 4-BPCA.