Microalgae are third generation feedstocks for bio-hydrogen production to achieve a low carbon economy. Nevertheless, the bio-hydrogen production from microalgae is generally low. In this study, an innovative free ammonia (FA, i.e., NH 3 ) pretreatment technology was first demonstrated to improve bio-hydrogen production from the secondary effluent cultivated microalgae during the anaerobic dark fermentation experiments. Scanning electron microscopy revealed that FA pretreatment disrupted microalgae surface morphology. The soluble chemical oxygen demand (SCOD) release increased from 0.01 g SCOD/g VS microalgae (VS = volatile solids) to 0.05-0.07 g SCOD/g VS microalgae after FA pretreatment of 240-530 mg NH 3 -N/L for 1 day, indicating the enhanced microalgae solubilization. Dark fermentation bio-hydrogen potential experiments showed that bio-hydrogen production from microalgae was substantially improved following FA pretreatment of 240-530 mg NH 3 -N/L. The bio-hydrogen production potential and maximum bio-hydrogen production rate increased from 18.2 L H 2 /kg VS microalgae and 2.5 L H 2 /kg VS microalgae/d to 19.9-22.1 L H 2 /kg VS microalgae and 3.1-3.8 L H 2 /kg VS microalgae/d, respectively, after FA pretreatment of 240-530 mg NH 3 -N/L was implemented on the microalgae for 1 day. This FA technology follows a circular economic model because the required FA is from the FA rich dark fermentation liquid, which is a wastewater treatment waste.