Abstract The effect of chitosan (CHI) on the stability of monodisperse modified lecithin (ML) stabilized soybean oil-in-water (O/W) emulsion was investigated. Monodisperse emulsion droplets with particle size of 24.4 ± 0.7 μm and coefficient of variation below 12% were prepared by microchannel (MC) emulsification using a hydrophilic asymmetric straight-through MC silicon 24 × 24 mm microchip consisting of 23,348 microchannels. The stability of the ML stabilized monodisperse emulsion droplets was investigated as a function of CHI addition at various concentration, pH, ionic strength, thermal treatment and freezing-thawing treatment by means of particle size and ζ-potential measurements as well as microscopic observation. The monodisperse O/W emulsions were diluted with CHI solution at various concentrations to a final droplet concentration of 1 wt% soybean oil, 0.25 wt% ML and 0–0.5 wt% CHI at pH 3. Pronounced droplet aggregation was observed when CHI was present at a concentration range of between 0.01 and 0.04 wt%. Above this concentration range, flocculations were less extensive, indicating some restabilization. ML stabilized emulsions were stable at a wide range of NaCl concentrations (0–1000 mM) and pH (3–8). On the contrary, in the presence of CHI, aggregation of the emulsion droplets was observed when NaCl concentration was above 200 mM and when the pH started to approach the pKa of CHI (i.e. ∼6.2–7.0). Emulsions containing CHI were found to have better stability at high temperature (>70 °C) in comparison to the emulsion stabilized only by ML. With sucrose/sorbitol as cryoprotectant aids, emulsions with the addition of CHI were found to be more resistant to droplet coalescence as compared to those without CHI after freezing at −20 °C for 22 h and thawing at 30 °C for 2 h. The use of CHI may potentially destabilize ML-stabilized O/W emulsions but its stability can be enhanced by selectively choosing the appropriate CHI concentrations and conditions of preparation.