Reduced irrigation in combination with biochar application can improve stomatal anatomy, water relations and intrinsic water use efficiency (WUEi), thus having a positive effect on the alleviation of salinity and drought stresses. A split-root pot experiment was executed to explore the effects of two biochar applications (WSP: wheat straw biochar; SWP: soft wood biochar) in combination with three irrigation strategies (FI: full irrigation; DI: deficit irrigation, PRD: partial root-zone drying irrigation) on stomatal anatomy, water relations and WUEi of cotton plants under normal soil (S0, EC=0.36 dS m-1) and saline soil (S1, EC=16.55 dS m-1). The results revealed that both salinity and drought stresses negatively affected plant water relations and reduced stomatal conductance, carbon isotope discrimination (Δ13C), stomatal size (SS) and hydraulic conductance (Kl) while increased leaf abscisic acid (ABA) concentration ([ABA]leaf). However, biochar amendment under salt stress significantly decreased [ABA]leaf while improved leaf water relations, increased Kl, stomatal density (SD), SS, Δ13C and maximum stomatal conductance (gsmax). Meanwhile, compared with FI and DI, PRD plants had greater SD, gsmax, and WUEi but lowered stomatal conductance (gs). Among all treatments, the combined application of WSP and PRD significantly increased SD and gsmax, improved leaf water relations, Kl and [ABA]leaf and WUEi. It is concluded that the altered stomatal features caused by the biochar and irrigation treatments are associated with changes in [ABA]leaf and Kl, and have a major role in affecting plant hydraulic integrity and water use efficiency of cotton exposed to salinity stress.