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Knee joint infection following total knee arthroplasty (TKA) is a serious condition and the treatments are complicated. The intra-articular solvent exchange-induced in situ forming matrix is of interest for modulating the release of antibiotics with a high drug concentration and a long period of exposed time at the target site. Stearic acid (S) and lauric acid (L) at various ratios were used as matrix formers by dissolving them in biocompatible solvents such as N -methyl pyrrolidone (NMP) and dimethyl sulfoxide (DMSO). Their matrix formation behaviors in phosphate buffer (pH 7.4) and hyaluronic acid (HA) solution were evaluated. Also, the density, viscosity, injectability, solvent diffusion, in vitro degradability and drug release using the dialysis tube method were investigated. The L:S ratio of 1:1 in DMSO exhibited rapid matrix formation and a remarkably low viscosity (7.67 ± 0.03 cp) with acceptable injectability (0.608 ± 0.027 N and 0.867 ± 0.010 N through 18-G and 27-G, respectively). Vancomycin HCl (V)-loaded L/S in situ forming matrix still provided ease of injection (1.079 ± 0.215 N and 1.230 ± 0.145 N through 18-G and 27-G needle, respectively) with fatty acid matrix formation after solvent exchange within 1 min, whilst V sustainably released over 6 days. It also presented effective antimicrobial activities against standard Staphylococcus aureus and methicillin-resistant Staphylococcus aureus strains. Therefore, V-loaded solvent exchange-induced in situ forming matrix using L and S as the matrix formers may be a potential local delivery system for treating knee joint infections occurring after TKA in the future. Unlabelled Image • Saturated fatty acids are of interest for modulating drug release with intra-articular solvent exchange-induced in situ forming matrix. • Vancomycin hydrochloride (V)-loaded stearic acid (S)/lauric acid (L) in situ forming matrix was developed • It had fast transformation into matrix after expose to phosphate buffer (pH 7.4) and hyaluronic acid (HA) solution • It exhibited effective antimicrobial activities especially for methicillin-resistant Staphylococcus aureus and sustained drug release over 6 days • V-loaded 40% 1:1 L:S in situ forming matrix exhibited the potential intra-articular drug delivery system [ABSTRACT FROM AUTHOR]