The molecular emission of BO and BO₂ radicals in laser-induced plasma has been investigated to analyze the boron isotopes in aqueous boric acid solution. The double-pulse laser configuration was employed to improve the detection sensitivity. The behaviors of molecular emissions were characterized depending on the interaction of sequentially configured lasers. Under the optimized conditions, the intensity of boron molecular emissions was significantly enhanced by about 10 times in comparison to that of the single-pulse configuration. The detection limit of boron in the aqueous solution was determined to be 72 ± 5 ppm. Furthermore, the isotopic shift between ¹⁰B and ¹¹B was observed at an individual rotational branch head. By utilizing these molecular emission spectra, the quantitative analysis of the isotopic ratio was conducted with partial least squares regression. In the cross-validation procedure, superior accuracy and precision were obtained. The root mean square error of prediction was 1.4% in the optimized spectral range. This study supports the understanding of molecular radical behavior according to the interaction of two sequential laser pulses and identifies the feasibility of molecular emission for real-time boron isotope detection in aqueous samples.