Isolation of circulating tumor cells from human blood plays a significant role in diagnosis and treatment of cancer. The most effective way of isolating cells is the use of lab on-chip microfluidic devices. This paper presents the development of cyclic olefin polymer membrane with an array of micropores for separating circulating tumour cells using ultrashort laser ablation process. Initially, the substrate is tested for its cell viability. The laser Fluence is varied from 1–12 J/cm2 and its observed that at a fluence of 2.5 J/cm2 the desired diameter of 12 μm with ablation depth of 1 μm is obtained which is necessary for CTC separation. The treated and untreated structure is characterized using Fourier transform infrared spectrometer and it shows that the treated region shows no significant shift in the spectrum after femtosecond laser ablation. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.