To improve the fluid mixing effect under micro-nano scale condition, we designed and manufactured a novel micro mixer with three-dimensional step structure based on the polymethyl methacrylate (PMMA) substrate material. The mixer was composed by one T-shaped inlet channel and six three dimensional step mixing units. The mixer was fabricated by using ultra-precision engraving machine and bonded by miscible organic solvents bonding method at the atmospheric pressure and low temperature conditions. Then, a series of numerical simulations were executed to optimize the step structures and investigate the mixing efficiency. The results showed that, when the Reynolds number was between 0.1 and 5, the concentration variance of mixing chamber cross section was less than 0.1. The reagent was close to the uniform mixture. When the flow rate within 1~20 mL/h, the mixer can achieve on precise regulation of solution pH. The micro machining technology and miscible organic solvents bonding method which were used for fabricate the 3D step micromixer are convenient and cost-effective. The mixer was conducive to the further promotion and application of microfluidic chips.