AbstractWe report a spore‐based biocatalysis platform capable of producing and self‐assembling active multimeric enzymes on a spore surface with a high loading density. This was achieved by co‐expressing both a spore surface‐anchoring scaffoldin protein containing multiple cohesin domains and a dockerin‐tagged enzyme of interest in the mother cell compartment during Bacillus subtilis sporulation. Using this method, tetrameric β‐galactosidase was successfully displayed on the spore surface with a loading density of 1.4 × 104 active enzymes per spore particle. The resulting spore biocatalysts exhibited high conversion rates of transgalactosylation in water/organic emulsions. With easy manufacture, enhanced thermostability, excellent reusability, and long‐term storage stability at ambient temperature, this approach holds a great potential in a wide range of biocatalysis applications especially involving organic phases.