This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9 K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350 U/mL under the optimal culture conditions of a 500 mL shaking flask containing 20 mL culture medium with the addition of 1.2 % (w/v) methanol, cultivation for 144 h at pH 7.0 and 27 °C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600 U/mL, 6,000 U/mL and 4,800 U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000 U/mL after 130 h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500 U/mL after 145 h incubation when methanol was used as the sole carbon source.