The purpose of this study was to investigate the influence of injection site on danofloxacin pharmacokinetics in Pekin ducks. Thirty-two male Peking ducks were divided into 4 equal groups as intravenous (IV), pectoral muscles (IMP), thigh muscles (IMT) and subcutaneous (SC). Danofloxacin was administered to all groups at a dose of 10 mg/kg. Plasma concentrations of danofloxacin were quantified by HPLC-UV. The volume of distribution at steady state and total clearance values of danofloxacin after IV injections in ducks were 4.43 L/kg and 0.42 L/h/kg, respectively. The terminal elimination half-life (t1/2ʎz), area under the concentration-versus time curve (AUC)0-last, and peak plasma concentration (Cmax) were similar after SC and IMP injections of danofloxacin. However, t1/2ʎz was shorter and AUC0-last and Cmax were lower in the IMT group compared to the IMP group. The bioavailability was lower in the IMT group than in the IMP and SC groups. Danofloxacin exhibited similar pharmacokinetic profiles after SC and IMP injection into Pekin ducks. In the IMT group, pharmacokinetics altered significantly, resulting in reduced body exposure and residence time of danofloxacin. Optimizing the injection site to subcutaneous or pectoral muscle routes may improve danofloxacin's therapeutic efficacy in Pekin ducks, while caution should be applied with thigh muscle injections due to reduced bioavailability and systemic exposure.