Enzyme-based antifouling coatings are potential alternative to traditional tributyltin-based coatings in the marine biofouling control depended on its environmental friendliness. Proteases are usually the key antifouling active components in enzyme based antifouling coatings. In this work, based on biological antagonism effect, a marine proteolytic bacterial strain of Bacillus velezensis was isolated from the sea mud, and denoted as SM-1. The scanning electron microscope (SEM) revealed that the bacteria are rod-shaped with length 1–1.3 μm. The protease-producing conditions of SM-1 were investigated, and it was found that the culture solution displays higher proteolytic activity under the culture conditions of 35°C, 10‰–20‰ salinity, pH 6–9 and more than 7 d culture time. Moreover, the 25 kDa protein was confirmed to be the main active component in the crude protease, which was revealed via the experiment of SDS-PAGE. The antifouling assay indicated that the protease SM-1 has remarkable effect on the decomposition of barnacle cement and diatom secretion, and also can effectively inhibit the attachment of barnacle cyprids, diatom and mussel byssus. Therefore, this protease potentially can be used as environmental-friendly antifoulant of enzyme-based marine antifouling coatings, and this work also provides a new approach to obtain antifouling protease via isolating proteolytic bacteria from the sea mud surrounding representative fouling organisms.