The creation of microporous surface modification of chitosan thin films irradiated by ultrashort laser pulses are studied. For this purpose, chitosan substrates were treated by using an amplified Ti:sapphire laser system at 800 nm central wavelength with 30 fs and 150 fs pulse duration and repetition rate 1 kHz and 50 Hz, respectively. Formation of surface modifications for both cases (30 fs and 150 fs) after femtosecond laser irradiation were observed. The threshold values for single-pulse (N = 1) and multi-pulse (N > 1) modification were evaluated by studying the linear relationship between the squared crater diameter D2 and the logarithm of the laser fluence (F) for N = 1, 2, 5, 10, 20, 30 and 50 number of laser pulses. The coefficient of incubation ξ, a major parameter in the process of surface modification and ablation of materials also was calculated for multi - pulse fluence threshold estimation by power - law relationship Fth (N) = Fth (1) Nξ-1, where N is the number of applied laser pulses. The surface properties of chitosan based thin films before and after femtosecond laser irradiation were investigated. The aim of this work is to determine the optimal morphological characteristics of the created structures for tailoring of protein adsorption and cell behavior.