Recently, the single-celled green freshwater microalgae species “Chlorella vulgaris” has attracted the attention of researchers due to its different usage areas. In particular, research focuses on the technology of obtaining bio‑hydrogen with various techniques. This research involves, for the first time, the use of the microalga Chlorella vulgaris as a bio-supporting material for magnetite Fe3O4 nanoparticles (Fe3O4NPs@Chlorella vulgaris) and the production of hydrogen through catalytic hydrolysis of NaBH4 (sodium borohydride, SB) in the presence of the resulting magnetite nanoparticles. Here, detailed kinetic studies were carried out during the SB-hydrolysis by taking magnetite Fe3O4NPs@Chlorella vulgaris and SB in varying amounts and at varying temperatures, and the activation energy and lifetime of magnetite Fe3O4NPs@Chlorella vulgaris was found to be 23.49 kJ mol−1 and 93,280 mol H2 (mol Fe3O4)−1, respectively. No change in the chemical and physical structure of the biocatalyst was observed during the hydrolysis of SB, so only detailed characterization of microalgae and magnetite Fe3O4NPs@Chlorella vulgaris was performed, and the particle size of the catalyst was calculated as 10.19 ± 2.17 nm. The results showed that these Fe3O4NPs@Chlorella vulgaris, which can be easily separated magnetically and have high catalytic activity, are a “clean” and quite surprising catalyst in terms of hydrogen production.