Surface modification of polydimethylsiloxane (PDMS) for organ‐on‐a‐chip (OOC) systems is fundamental for the success of cell physiological assays. Although UV light is commonly used for this purpose, surface chemical modifications are only temporary. To overcome these limitations, an alternative approach is proposed: a physicochemical modification using ozone and the heterofunctional crosslinker sulfo‐SANPAH (SS). This simple one‐step approach is carried out on PDMS microchannels of OOC platforms. A broad physicochemical characterization based on spectroscopy and microscopy techniques confirms successful modification of the PDMS surface. The PDMS surface contact angle decreases after addition of SS (from 101.4° to 79.9°) but decreases as low as 20.8° after extracellular matrix deposition, demonstrating successful transition of the surface from hydrophobic to hydrophilic. For biological characterization, human liver sinusoidal endothelial cells (LSEC) and murine primary hepatocytes are cultured in OOC channels for up to 7 days. LSECs and hepatocytes establish strong cell adhesions under laminar flow and form a well‐established canaliculi network, respectively. This one‐step method of PDMS surface modification for OOC is simpler than others reported in the literature (e.g., based on 3‐aminopropyltriethoxysilane) and ideal for beginners interested in using microfluidic platforms for cell culture.