The invention discloses an absorbancy-based high-accuracy nucleic acid concentration determining method. The method comprises the following steps: (1) preparing a nucleic acid standard substance, a nucleic acid sample and a corresponding blank buffer solution; (2) using the blank buffer solution for performing gradient dilution on the nucleic acid standard substance, respectively determining the absorbancy of the standard substance at each concentration level and a to-be-tested sample after the dilution and calculating initially tested concentration c0 of the sample; (3) using the nucleic acid standard substance for preparing a pair of standard substances K1c0 and K2c0 according to the initially tested concentration, adopting a bracketing method for further determining the concentration of the nucleic acid sample, calculating respective standard uncertain degree mu1 and mu2 of the pair of standard substances according to a preparation process and further calculating a compounding standard uncertain degree uc of a determined result; (4) solving a nonlinear plan and minimizing the uncertain degree, thereby acquiring specific values of K1 and K2; (5) preparing high standard and low standard according to the calculated values of K1 and K2 and adopting the bracketing method for determining the sample, thereby acquiring an accurate result c of the sample.