Surfaces with gradient properties are of central importance for a number of chemical and biological processes. Here, we report rapid generation of a polydopamine (PDA) gradient on hydrophobic surfaces by a simple, low cost, and general technology, cyclic draining-replenishing (CDR). Due to the unique surface chemistry of PDA, it enables continuous and precise control of surface wettability and subsequent deposition of organic and inorganic compounds. Using kanamycin as a model compound, we show that the gradient PDA membrane potentially can be used to prepare minimum inhibitory concentration (MIC) test strips for quantifying resistance of antimicrobial agents from microorganisms. Because CDR is experimentally simple, scalable, fast, and does not require specialized reagents or instruments, we envision this platform can be easily adopted to create a variety of functional surfaces.