This paper presents the fabrication of miniaturized microfluidic device based on cyclic olefin polymer for the separation of serum from blood. The characteristics and performance of the proposed micro-holes array are analyzed for its trapping of red blood cell, serum separation and cholesterol presence using FEM numerical simulation. An array of micro-holes are created on biocompatible cyclic olefin polymer using femtosecond laser ablation. A single track of experiments has been carried out to obtain the required depth of 2microns with a diameter ranging from (8-27)µm for various Fluence levels of the incident laser beam. The surface morphology is characterized using optical microscopy images. The fabricated device is tested for its characteristics in real time in a clinical laboratory. The obtained results match well with the simulation results. © 2019 National Institute of Optoelectronics. All rights reserved.