Abstract <p>The industrial production of menaquinone-7 (MK-7) by <i>Bacillus subtilis</i> has been historically constrained by significant challenges in bioprocess efficiency. To address these limitations, we explored an innovative immobilization strategy utilizing a porous thin-film hydrogel system. Specifically, we developed a novel porous thin-film PVA + B@Ca hydrogel immobilization method that fundamentally transforms cell encapsulation and fermentation dynamics. The comparison between PVA + B@Ca hydrogel immobilized cells and free cells in fermentation demonstrated a significant increase in MK-7 yield from 32.76 ± 1.92 to 48.33 ± 2.92&#xa0;mg/L, as well as a reduction of the fermentation duration from 48 to 24&#xa0;h. Additionally, the immobilized cells demonstrated good stability during continuous fermentation, resulting in a space–time yield of MK-7 that increased to 2.0&#xa0;mg/L·h, which was five times higher than that achieved with free-cell fermentation. Mechanistic insights revealed through microscopic analysis highlight the transformative nature of the hydrogel immobilization: The PVA + B@Ca hydrogel’s porous structure creates a protective microenvironment that mitigates cellular stress and maintains optimal metabolic conditions. These findings represent a paradigm shift in understanding cellular immobilization, demonstrating how strategic encapsulation can fundamentally enhance MK-7 fermentation biotechnology.</p> Key points <p><i>• A novel hydrogel immobilization method was developed for MK- 7 production</i>.</p> <p><i>• The use of immobilized cells gave a fivefold improvement in the space–time yield</i>.</p>