Surface hydrophobicity of organisms provides biological self-protection. The hydrophobicity of pest surface, acting as a main obstacle for the pest control, can lead to low utilization and high loss of pesticides. Dermanyssus gallinae is a notorious pest in egg-laying hens, whose control primarily depends on acaricide spraying, while its surface hydrophobicity and potential influence on pesticide effectiveness are not clear. In the present study, the contact angle measurements revealed that the surface of D. gallinae was hydrophobic. Analysis using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the surface microstructures of D. gallinae consist of cuticular folds, with a lipid-rich outermost layer of the cuticle. Based on gas chromatography-mass spectrometry (GC-MS) and gas chromatography (GC), it was found that the major compositions of cuticular lipids were fatty acids and n-alkanes. Modifying the chemical compositions and microstructures of the D. gallinae surface resulted in a reduction in surface hydrophobicity and an increase in the permeation of Rhodamine B through the cuticle. This observation suggested that the chemical compositions and microstructures were pivotal in determining surface hydrophobicity, hindering compound penetration into the cuticle. Finally, it was found improving the wettability of pesticide solution by adding surfactants could overcome the surface hydrophobicity and enhance the efficacy of pesticide against the mites. This study sheds light on the surface hydrophobicity mechanism of D. gallinae and provides a novel strategy to improve the efficacy of acaricides against the mites.