Diversified members of hop bitter acids (alpha- and beta-acids) have been found in hop (Humulus lupulus). Mixtures of hop bitter acids have been traditionally applied in brewing and food industries as bitterness flavors or food additives. Recent studies have discovered novel applications of hop bitter acids and their derivatives in medicinal and pharmaceutical fields. The increasing demands of purified hop bitter acid promoted biosynthesis efforts for the heterologous biosynthesis of objective hop bitter acids by engineered microbial factories. In this study, the updated information of hop bitter acids and their representative application in brewing, food, and medicine fields are reviewed. We also speculate future trends on the development of robust microbial cell factories and biotechnologies for the biosynthesis of hop bitter acids.