In this research, we have developed and demonstrated a fabrication method for the formation of various shapes of silicon freestanding microfluidic channels and microstructures in one-step photolithography. The fabrication process utilizes the silicon direct wafer bonding with silicon nitride as an intermediate layer, local oxidation of the silicon (LOCOS) process and wet anisotropic etching. Two different types of etchants (non-ionic surfactant (Triton-X-100) added and pure 25 wt% TMAH solutions) are used in series to perform silicon anisotropic etching. Surfactant-added tetramethyl ammonium hydroxide (TMAH) is employed to define the shapes of the structures, while pure TMAH is used to get high undercutting for their fast releasing. The non-ionic surfactant is preferred considering the complementary metal-oxide semiconductor (CMOS) post process issue of wet anisotropic etching. The undercutting at sharp and rounded concave corners, edges aligned along 〈1 0 0〉 directions, is measured and analyzed in both pure and surfactant-added TMAH solutions. Mask design issues that must be taken into consideration for the fabrication of desired shape and size structures are also presented. © 2009 IOP Publishing Ltd.