β-chitosan preparation from squid pens was carried out using aqueous NaOH with the ultrasonic assistance. Single factor experiments and L9 (34) orthogonal experiments were used to investigate the effect of three parameters (reaction time, concentration of NaOH and reaction temperature) on deacetylation of β-chitin. The optimal conditions for deacetylation of chitin were reaction temperature 80°C, reaction time 2 h and concentration of NaOH 50%. The optimal conditions allowed deacetylation degree of β-chitin from 71.32% to 92.91%. The β-chitosan from squid pens was confirmed by Fourier transform infrared spectroscopy. The antibacterial activities of the prepared β-chitosans again staphylococcus aureus (S. aureus) and Escherchia coli (E.coli) were then determined and compared by the MIC (minimum inhibitory concentration). Results indicate that β-chitosans with different degrees of deacetylation (DD) possess different antibacterial activity. The growth of S. aureus can be easily inhibited by prepared β-chitosan than E.coli.