Objective: To study the otoscopic, tympanometric changes and curative effect of intranasal pulmonary surfactant (PS) drops on secretory otitis media (OME) in guinea pigs. Methods: An OME model was established by injecting Haemophilus influenzae solution into the tympanic cavity. Ten animals were assigned as the normal control group, 10 as the model control group, 10 as the saline treatment group, and 11 as the PS treatment group. Changes in the tympanic membrane and middle ear pressure were recorded via otoscopy and tympanometry. Results: The tympanogram was type-A in the normal group and this changed to type-C in the model-control group and in the saline-treated group. The tympanogram changed to type-B in the PS-treated group. Statistically significant differences in middle ear pressure and peak acoustic admittance were observed among all groups except between the model control and saline treatment groups. Conclusion: The study demonstrated that administration of intranasal pulmonary surfactant drops plays a significant role in reducing effusion and correcting negative middle ear pressure in guinea pigs with otitis media with effusion.