In vivo electrochemistry with a carbon-fiber electrode (CFE) is the most useful method for tracking neurochemicals in specific brain regions due to its high spatiotemporal resolution. However, CFE is inevitably subject to surface biofouling that leads to a decrease in sensitivity. Here, we develop a polytannic acid (PTA)-doped nanoporous conductive polyaniline (PANI) membrane-coated CFE to minimize biofouling-induced negative effects for in vivo analysis. The as-prepared PTA–PANI-coated CFE shows excellent antifouling property and enrichment capacity toward electrochemical measurement of dopamine (DA) in physiological pH. The PTA–PANI-coated CFE can in vivo monitor the release of DA induced by electrical stimulation and exhibits almost the same sensitivity in the postcalibration (S post) and the precalibration (S pre; S post/S pre = 0.90). We believe this conductive nanoporous membrane-coated CFE offers a new platform for in vivo measurement, which would help probe brain chemistry.