Bottom-up tissue engineering provides a promising way to recreate complex structural organizations of native organs in artificial constructs by assembling functional repeating modules. However, it is challenging for current bottom-up strategies to simultaneously produce a controllable and immediately perfusable microfluidic network in modularly assembled 3D constructs. Here we presented a bottom-up strategy to produce perfusable microchannels in 3D hydrogels by sequentially assembling microfluidic modules. The effects of agarose-collagen composition on microchannel replication and 3D assembly of hydrogel modules were investigated. The unique property of predefined microchannels in transporting fluids within 3D assemblies was evaluated. Endothelial cells were incorporated into the microfluidic network of 3D hydrogels for dynamic culture in a house-made bioreactor system. The results indicated that the sequential assembly method could produce interconnected 3D predefined microfluidic networks in optimized agarose-collagen hydrogels, which were fully perfusable and successfully functioned as fluid pathways to facilitate the spreading of endothelial cells. We envision that the presented method could be potentially used to engineer 3D vascularized parenchymal constructs by encapsulating primary cells in bulk hydrogels and incorporating endothelial cells in predefined microchannels.