Objective To study the correlation of azithromycin niosomes (AN) in vitro release with its in vivo absorption. Methods Laser particle size analyzer was used to measure the particle size and zeta potential of AN. An UV method was established to analyze the in vitro release of AN; HPLC method was used to determine the plasma concentration of azithromycin after a single oral dose of drugs in rats. Then Loo-Riegelman and deconvolution method were used to calculate the absorption fraction (Fa) and input function (R). Results The AN size was (907.4 ± 0.10) nm and its zeta potential was (14.6 ± 0.02) mV. The regression equation of peak area ratio of AN to internal standard (A) and the concentration of azithromycin (C) was: A= 19.26C-52.69, r=0.999 0 (n=3); the in vitro regression equation determined at 482 nm was: D= 0.014 4C-0.070 1, r=0.999 6 (n=3); and the recoveries all met the requirements. If Y was cumulative in vitro release and Fa was the absorption percentage, the regression equation was: Fa=0.981 8Y + 1.083 1, r=0.956 5; if Y was the cumulative in vitro release and R was the input function, the regression equation was: R=0.763 5Y-15.69 9,r=0.862 7. Conclusion There is no correlation between azithromycin in vitro release with its in vivo absorption.