This study presents a potential approach to enhance integrated sequential biohydrogen production from waste biomass using magnetite nanoparticle (Fe3O4 NPs) which is & nbsp;synthesized through waste seeds of Syzygium cumini. Consequences of 0.5% Fe3O4 NPs have been investigated on the thermal and pH stability of fungal crude cellulase. It is noticed that Fe3O4 NPs treated enzyme and control exhibits 100% activity in the temperature range of 45-60 degrees C and 45-55 degrees C, respectively. Moreover, Fe3O4 NPs treated enzyme showed extended thermal stability in the temperature range of 50-60 degrees C up to 12 h. Beside this, Fe3O4 NPs treated enzyme possesses 100% stability in the pH range of 5.0-7.0 whereas control exhibited only at pH 6.0. Enzymatic hydrolysis via Fe3O4 NPs treated enzyme has been employed which produces-68.0 g/L reducing sugars from sugarcane bagasse. Sub-sequently, sugar hydrolyzate has been utilized as substrate in the sequential integrated fermentation that produces-3427.0 mL/L cumulative hydrogen after 408 h. This approach may have potential for the pilot scale production of biohydrogen from waste biomass at low-cost in an eco-friendly manner. (c) 2021 Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.