Hypothesis: Microfluidic technology plays a significant role in separating biomolecules, because of its miniaturization, integration, and automation. Introducing micro/nanostructured functional materials can improve the properties of microfluidic devices, and extend their application. Inverse opal has a three-dimensional ordered net-like structure. It possesses a large surface area and exhibits good mass transport, making it a good candidate for bio-separation. This study exploits inverse opal titanium dioxide-zirconium dioxide films for on-chip phosphopeptide enrichment. Experiments: Titanium dioxide-zirconium dioxide inverse opal film-based microfluidic devices were constructed from templates of 270-, 340-, and 370-nm-diameter poly(methylmethacrylate) spheres. The phosphopeptide enrichments of these devices were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Findings: The device constructed from the 270-nm-diameter sphere template exhibited good comprehensive phosphopeptide enrichment, and was the best among these three devices. Because the size of opal template used in construction was the smallest, the inverse opal film therefore had the smallest pore sizes and the largest surface area. Enrichment by this device was also better than those of similar devices based on nanoparticle films and single component films. The titanium dioxide-zirconium dioxide inverse opal film-based device provides a promising approach for the efficient separation of various biomolecules. (C) 2016 Elsevier Inc. All rights reserved.