Currently, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development, and the agarases from marine bacteria become one of the crucial enzymes that can hydrolyze the marine biomass. In this research, a recombinant agarase, AgaXa, was fermented by the gene-engineered E. coli strain, and the process was optimized to increase its production. Linear fed-batch cultivation within a 5 L bioreactor (25 °C and 0.1 mM IPTG induction) resulted in the highest production of 1.5×104 U/L, a six-fold improvement over the non-optimized conditions. For further industrial applications, the optimal protective reagent system to maintain the enzymatic thermostability were determined by adding Na+ (1.8 mM), glucose (1.37%), glycerol (16.97%) and pectin (0.21%). Under such optimal conditions, more than 80% of agarase activity was retained even when the agarase was incubated at 85 °C. In order to apply the fermented agarase to convert agar polysaccharide, galactose, the main component of agar, was firstly investigated on its potential of biohydrogen production by using Enterobacter sp. strain CN1, and the highest hydrogen yield of 303.2 mL/g was obtained within the cultivation media containing 5.87 g/L of galactose, together with an initial pH of 7.3 and incubation temperature of 36 °C. Via the synergetic saccharification process using AgaXa, together with another neoagarobiose hydrolase (NH852), the obtained agar hydrolysate was further applied to generate biohydrogen by strain CN1, and the biohydrogen production (5047±228 mL/L) was detected from 50 g/L of agar, demonstrating a 3.86-fold higher production rather than the control without the pretreatment of agarases. This work for the first time reveals a systematic investigation on the fermentation of agarase for the production of biohydrogen from agar polysaccharides, and it also provides the positive impacts on the sustainable utilization of marine biomass for bioenergy generation.