In weak acidic medium, bisphenylnaphthylmethane dyes, such as night blue (NB),Victoria blue B (VBB) and Victoria blue 4R (VB4R) , react rapidly with sodium hyaluronate to form complexes, which causes the change of absorption spectra, the decrease of absorbance near 612nm(NB),616nm(VBB),585nm(VB4R), separately, and ΔA(=A-A0) values are proportional to the concentration of sodium hyaluronate, Beer’s law is obeyed in the range between 0.5~2.0 mg/L(NB) , 0.1~2.5mg/L(VBB) and 0.1~0.6 mg/L(VB4R) for sodium hyaluronate. The molar absorptivities of different systems are between 1.51×108~ 5.52×108 (the molecular weight of SH is 5×105). Based on these, we developed a new spectrophotometric method for the determination of sodium hyaluronate. The method is simple, rapid, repeatable, and it has good selectivity. It has been used for the determination of total amounts of sodium hyaluronate in samples with satisfactory results.