There is an urgent demand to construct multiplexed biomolecular patterns to obtain more biological information from a single experiment. However, with only limited reports focusing on defective top-down approaches, challenges remain to develop a bottom-up strategy for multiplexed patterning. To this end, a novel strategy has been proposed to fabricate multiplexed DNA patterns via macroscopic assembly through combined precise magnetic manipulation and DNA hybridization-driven self-assembly. Therefore, a multiplexed DNA pattern composed of glass fibers loaded with multiple specific strands of DNA was constructed, and its potential application in simultaneous detection of multiplex target DNA was demonstrated. Moreover, the fabricated multiplexed DNA pattern shows an erasable behavior because the hybridized DNA can be disassembled by strand displacement.