Pseudomonas aeruginosa keratitis (or pink eye) is a challenging ocular infection that causes serious complications due to the deficiency of effective antibiotic treatment. Thus, in this study we isolated and characterized a specific bacteriophage, phage vB_Pa_ZCPS1, to be used to formulate an in situ- gel loaded bacteriophage for an in vivo rabbit infection treatment model. Phage vB_Pa_ZCPS1 is a double-stranded DNA bacterial virus, of 46,135 bp encoding 75 open reading frames (ORFs) with no antibiotic resistance genes detected. Moreover, it has a podoviral morphotype from the Caudoviricetes class with a 62.4 nm capsid and a short inflexible tail of around 18.8 nm, as indicated by the transmission electron microscope (TEM). Phage vB_Pa_ZCPS1 presented good stability to the UV exposure and a wide range of pH values from 3.0 to 11.0. In addition, the phage-bacteria dynamics study showed that phage vB_Pa_ZCPS1 was effective against P. aeruginosa, especially at low multiplicities of infections (MOIs), including 0.001, 0.01, and 0.1. Respectively, it was loaded to the characterized in situ gel composed of 14 % Pluronic F-127 and 1.5 % HPMC K4M polymer. The in situ-gel has a gelling time of 30 s ± 1, and a temperature of 33 °C ± 1, where the viscosity of the gel increases 10-fold. For the in vivo trial, the infected group treated with phage presented improved clinical outcomes, where the histopathological analysis revealed normal corneal thickness and intact corneal stratified squamous epithelium. Thus, the in situ-gel loaded phage vB_Pa_ZCPS1 could be a potential candidate approach to treat P. aeruginosa keratitis.