Florfenicol (FF) is a novel animal-specific amidohydrin broad-spectrum antibiotic. However, its aqueous solubility is extremely poor, far below the effective dose required for veterinary clinic. Thus, FF is often used in large doses, which significantly limits its preparation and application. To overcome these shortcomings, the FF-hydroxypropyl-β-cyclodextrin (FF-HP-β-CD) inclusion complexes were developed using the solution-stirring method. The physical properties of FF-HP-β-CD were characterized. A comparison was conducted between FF and FF-HP-β-CD freeze-dried powder injection of their muscle irritation and the pharmacokinetics. The drug loading and saturated solubility of FF-HP-β-CD at 37 °C were 11.78% ± 0.04% and 78.93 ± 0.42 mg/mL, respectively (35.4-fold compared with FF). Results of scanning electron microscopy, differential scanning calorimetry, X-ray diffraction, and Fourier transform infrared showed that FF was entrapped in the inner cavity of HP-β-CD, and the inclusion complex formed in an amorphous state. In comparison with FF commercial injection, FF-HP-β-CD increased the elimination half-life (t1/2β), transport rate constant (K10, K12, K21), and maximum concentration (Cmax) after intramuscular injection in beagle dogs. Conversely, it decreased the distribution half-life (t1/2α), absorption rate constant (Ka), apparent volume of distribution (V1/F), and peak time (Tmax). These results suggest that FF-HP-β-CD freeze-dried powder injection is a promising formulation for clinical application.