A novel technique of polymer-based monolithic high performance liquid chromatography (HPLC) is proposed for peptides and proteins separation on chip. The cyclic olefin copolymer (COC) chip consists of the two injection channels for gradient elution delivery, two injection channels for sample injection and waste, and one spiral separation channel regulated with “double-T” cross-injector. This device is integrated with 4 embedded needles at reservoirs which are located at the end of injection channel for connecting programmable syringe pumps and shut-off valves to control sample injection and elution. After hot-embossed process to imprint channel geometric pattern, the chip is bonded by cyclohexane at room temperature without sacrificial material or other complicated processes which may cause contaminate or decrease yield production. The needles are inserted in suitable size reservoirs followed with annealing to eliminate residual stress and shear stress. The needle interface could connect with union which is easy to integrate with capillary, pump and valve. It could withstand of pressure as high as 24.8Mpa (244.76 atm) without crack observed on the chip. The UV-initiated grafting COC microfluidic chips followed by polymerization of porous poly(butyl methacrylate-co-ethylene dimethacrylate) monolith has achieved protein and peptide separation with gradient eluting. The gradient is generated by simultaneously injecting low and high concentration organic solvents with different injection velocities through a nanoliter mixer with optimized grooved structures located at the converging of 2 injection channels. Preliminary separation tests show that peptide and protein mixtures were well separated in 30mins. The polymer-based chip could operate 5 days for continuous separation test without delamination or cracks.