Lysozyme is an antimicrobial compound, which has been used in both food and pharmaceutical industry. Kluyveromyces lactis K7 is a genetically modified microorganism, which is proposed to produce the human lysozyme commercially. Up to now, human lysozyme production was enhanced by the application of biofilm reactor with plastic composite (173 U/ml) compared to the suspended-cell bioreactor (110 U/ml). However, lysozyme has antimicrobial peptides, which might affect the growth of yeast cells during fermentation. As a result, high production potential of human lysozyme might be reduced. Therefore, a simultaneous fermentation and online recovery system can help to enhance the production. In order to test this strategy, the objective of this study was to select the best adsorption and desorption conditions for the resin, silicic acid, and evaluate the simultaneous fermentation and online recovery system for the production of human lysozyme by K. lactis K7 in biofilm reactor. The results demonstrated that the best adsorption (95.6% adsorption) was obtained at 25°C, pH 4, and 25% silicic acid ratio. Also, the best desorption parameters were determined as 25°C, pH -6.2, and 5% sodium dodecyl sulfate with 1 M NaCI and 20% ethanol as eluent composition. Four repetitive desorption process provided up to 98% desorption. Finally, the simultaneous fermentation and online recovery system with tangential flow microfilter module improved the production of human lysozyme to280.4 U/ml, which is 63% improvement compared to the biofilm bioreactor without online recovery system.