A simple and flexible approach is developed for controllable fabrication of spider-silk-like microfibers with tunable magnetic spindle-knots from biocompatible calcium alginate for controlled 3D assembly and water collection. Liquid jet templates with volatile oil drops containing magnetic Fe3O4 nanoparticles are generated from microfluidics for fabricating spider-silk-like microfibers. The structure of jet templates can be precisely adjusted by simply changing the flow rates to tailor the structures of the resultant spider-silk-like microfibers. The microfibers can be well manipulated by external magnetic fields for controllably moving, and patterning and assembling into different 2D and 3D structures. Moreover, the dehydrated spider-silk-like microfibers, with magnetic spindle-knots for collecting water drops, can be controllably assembled into spider-web-like structures for excellent water collection. These spider-silk-like microfibers are promising as functional building blocks for engineering complex 3D scaffolds for water collection, cell culture, and tissue engineering.