CYP1A is a heme-thiolate enzyme associated with the cytochrome P4501A1 monooxygenase system and is inducible by a wide variety of xenobiotics and endogenous ligands that bind and activate the aryl hydrocarbon receptor (AHR). The AHR-CYP1A axis is important for detoxification of certain xenobiotics and for homeostatic balance of endogenous sex hormones, amine hormones, vitamins, fatty acids, and phospholipids. Herein, we generated and described applications of a zebrafish CYP1A-targeted monoclonal antibody (mAb CRC4) that fortuitously recognizes induced CYP1A across vertebrate taxa, including fish, chicken, mouse, rat, and human. We then demonstrated that mAb CRC4 targets a highly conserved epitope signature of vertebrate CYP1A. The unique complimentary determining region (CDR) sequences of heavy and light chains were determined, and these Ig sequences will allow for the expression of recombinant mAb CRC4, thus superseding the need for long-term hybridoma maintenance. This antibody works well for immunohistochemistry (IHC), as well as whole-mounted IHC in zebrafish embryos. Monoclonal antibody CRC4 may be particularly useful for studying the AHR-CYP1A axis in multiple vertebrate species and within the context of Oceans and Human Health research. By using archived samples, when possible, we actively promoted efforts to reduce, replace, and refine studies involving live animals.