This paper is to study the release characteristics of doxycycline (DC) raw material drug and doxycycline chitosan nanoparticles (DC-CS-NPs) freeze-dried powder in vitro and in vivo by ultraviolet spectrophotometry and ultra performance liquid chromatography, respectively. Results showed that the release behaviors of DC raw material drug were simulated the most in the zero order dynamic equation in artificial gastric juice, artificial intestinal juice and pH 7.4 buffer phosphate. The release of DC raw material drug was fast which could completely dissolve in one hour; The DC-CS-NPs freeze-dried powder had a significant slow-release characteristic. The release behaviors were simulated the most in the biexponential equation. The drug released fast in the early stage while it released slowly later. The order of release speed of DC-CS-NPs freeze-dried powder from fast to slow was pH 2.0 artificial gastric juice, pH 3.0 artificial gastric juice, pH 4.0 artificial gastric juice, artificial intestinal juice and pH 7.4 buffer phosphate. When DC raw material drug became DC-CS-NPs freeze-dried powder, after a single oral administration at a dose of 20 mg/kg at (25±1) ℃, the double-peak phenomenon of plasma in channel catfish was turned into single-peak, and the pharmacokinetic parameters of DC-CS-NPs freeze-dried powder were changed. Compared to DC raw material drug, the peak concentration (Cmax) decreased. The peak time (Tmax) and terminal elimination half-life (T1/2β) prolonged. The area under concentration-time curve (AUC) was larger. The DC-CS-NPs freeze-dried powder will become an ideal DC new formulation.