Glyceric acid (GA), a carboxylic group-containing diol, is obtained from bioresources via microbial processes. In this study, we aimed to develop a reactive extraction method to separate GA from aqueous solutions using 2-naphthaleneboronic acid (2NB) and tri-octyl methyl ammonium chloride (TOMAC). Different feed molar amounts of 2NB (0–25 µmol), TOMAC (0–500 µmol), and NaOH (0–250 µmol) were used for GA (2.5 µmol) separation. A combination of 25 µmol 2NB, 100 µmol TOMAC, and 25 µmol NaOH was determined to be optimal for GA separation, providing 66.8 ± 3.2% GA yield at pH 11. GA was extracted by 2NB and TOMAC in a coordinated manner. Moreover, effects of various carboxylic acids (acetic, lactic, succinic, malic, tartaric, and citric acids) on GA separation from aqueous solutions were investigated. Interestingly, no significant effect on GA yeild (60.3 ± 1.2–65.2 ± 2.5%) was observed regardless of the type of carboxylic acid. The optimized protocol was subsequently applied to separate GA from crude GA solution prepared by incubating glycerol with the cells of the acetic acid bacterium, Acetobacter tropicalis NBRC 16470. GA separation was achieved at a comparable level (yield: 70.6 ± 4.6% and purity: 76.1 ± 4.1%) as that achieved using a GA reagent. This study demonstrated the efficiency of the repeated use of the organic phase for GA separation, with no significant changes in GA yield.