Lysozyme is a lytic enzyme, which has antimicrobial, antiviral, anti-inflammatory activities. Enhancement of lysozyme production is required to meet its current demand in pharmaceutical and food industry. Egg white lysozyme is the major commercial source of lysozyme. However, egg-white lysozyme poses immunological problems when applied to human beings and it has a four-times less specific activity than human lysozyme. There are are several approaches for human lysozyme production and microbial fermentation is one of them. Biofilm reactors with plastic composite support have been evaluated for human lysozyme production by fermentation of Kluyveromyces lactis K7 in our earlier study, which demonstrated significant improvement in batch fermentation of human lysozyme. Biofilm reactor with plastic composite support is a passive immobilization reactor, which includes attachment of cells onto support without using any chemical agent. Fed-batch mode includes a step to feed the reactor with additional carbon source during the fermentation process. Extra substrate availability in fed-batch bioreactors could provide higher production levels and yields and prevent substrate inhibition. Therefore, this study was undertaken to evaluate different carbon source addition times and different initial carbon sources and their concentrations to increase the human lysozyme production by K. lactis K7 in biofilm reactor in fed-batch fermentation. Result showed that addition of carbon source in the log phase (177 U/ml) than the late log phase (174 U/ml) provided higher lysozyme production. Moreover, human lysozyme production by K. lactis K7 with glucose as initial carbon source (187 U/ml) was found higher than the production just with lactose (178 U/ml).