The hydrophilic spindle-knot microfiber serves as a valuable tool for water collection to combat freshwater scarcity. However, balancing microfiber's water collection efficiency and reusability remains a challenge. To address this contradiction, a microfluidic approach utilizing enzyme-mediated, in situ biomineralization is developed to produce biomineralized rough spindle-knot calcium alginate microfibers (BSMs). Specifically, by strategically organizing enzymes within the microfiber, biomineralization occurs mostly at the knot, forming CaCO