The invention discloses a determination method for the relative abundance of boron isotope in boric acid. The method comprises the steps of: preparing a boric acid solution; adding a hydroxyl-containing compound into the boric acid solution to carry out reaction on boric acid and the hydroxyl-containing compound so as to form ester or chelate; and detecting the reacted solution by mass spectrum. According to the invention, the pretreatment reaction conditions are mild, the rate is fast, the ester or chelate obtained by reaction of boric acid and the hydroxyl-containing compound can be detected by electrospray ionization source equipped mass spectrum, the detection result is accurate, the whole detection cycle is very short, and the method can be used for determination of mass samples.