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.