A method for rapid screening and identification of 30 protein assimilation hormones and glucocorticoids in fish was established using ultra-high performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q/Orbitrap HRMS). The samples were extracted with 80% acetonitrile aqueous solution (containing 0.2% formic acid), centrifuged, purified by solid phase extraction (SPE) on Oasis PRiME HLB column, and dried with nitrogen blow. The residue was re-dissolved. The chromatographic separation was performed on a Waters Acquity BEH C₁₈ (2.1 mm × 100 mm, 1.7 μm) column through gradient elution using a mobile phase consisting of 20 mmol/L ammonium acetate aqueous solution containing 0.1% formic acid (A) and acetonitrile (B). The target analytes were detected in the positive and negative ion mode using a heated electrospray ionization (HESI) source through primary full scan/data-dependent secondary scan (full MS/dd-MS²) monitoring, and quantitated by matrix-matched external standard method. The results showed that good linearity was observed for the 30 hormones in the concentration range of 0.5–100 ng/mL with correlation coefficients (r) greater than 0.9950. The limits of detection were between 0.2 and 1.0 μg/kg, and the limits of quantification were between 0.5 and 2.0 μg/kg. The recoveries were 69.7%–103.2% with relative standard deviations (RSDs) of 2.3%–9.4% at three spiked levels for four different fishes (turbot, mandarin fish, mullet and grass carp). The proposed method is efficient, accurate and reliable, and is suitable for the rapid multi-objective screening and quantitative analysis of protein assimilation hormones and glucocorticoids in fish.