A method modifying a vacuum-assisted UV micro-molding (VAUM) process is proposed for the fabrication of polymer two-level submicron high porosity membranes (2LHPM). The modified process allows for the fabrication of robust, large-area membranes over 5 × 5 cm2 with a hierarchical architecture made from a 200 nm-thick layer having submicron level pores (as small as 500 nm) supported by a 20 μm-thick layer forming a microporous structure with 10-15 μm diameter pores. The fabricated freestanding membranes are flexible and mechanically robust enough for post manipulation and filtration of cell samples. Very high white blood cell (WBC) capture efficiencies (≈97%) from healthy blood samples after red blood cell (RBC) lysis are demonstrated using a 3D-printed filter cartridge incorporated within these 2LHPM. A high release efficiency of ≈95% is also proved using the same setup. Finally, on-filter multistep immunostaining of captured cells is also shown.