Abstract: | True three‐dimensionally (3D) integrated biochips are crucial for realizing high performance biochemical analysis and cell engineering, which remain ultimate challenges. In this paper, a new method termed hybrid femtosecond laser microfabrication which consists of successive subtractive (femtosecond laser‐assisted wet etching of glass) and additive (two‐photon polymerization of polymer) 3D microprocessing was proposed for realizing 3D “ship‐in‐a‐bottle” microchip. Such novel microchips were fabricated by integrating various 3D polymer micro/nanostructures into flexible 3D glass microfluidic channels. The high quality of microchips was ensured by quantitatively investigating the experimental processes containing “line‐to‐line” scanning mode, improved annealing temperature (645°C), increased prebaking time (18 h for 1mm‐length channel), optimal laser power (1.9 times larger than that on the surface) and longer developing time (6 times larger). The ship‐in‐a‐bottle biochips show high capabilities to provide simultaneous filtering and mixing with 87% efficiency in a shorter distance and on‐chip synthesis of ZnO microflower particles. |