Macleaya cordata is a traditional herb medicine with alkaloids as the main bioactive substance. To identify alkaloid compounds from M. cordata, its crude extract was obtained with 0.2 mol/L hydrochloric acid, and alkaloid compounds in the demineralised extract by organic solvents from crude extract were qualitatively and quantitatively analysed by UPLC-DAD-QTOF-MS/MS. Through systematic analysis of retention times, mass spectrometry data, and diagnostic fragmentation pathways and rules, 32 alkaloids were rapidly unambiguously identified or tentatively deduced by comparison with standard MS spectra or literature data. Among them, 16 minor alkaloid compounds including nandazurine, hydroxychelidonine, capauridine, (-)-dicentrine, leptopine, adlumidine, takatonine, (2, 3)-trans-N-(p-hydroxyphenethyl)ferulamide, 9-ethoxyaristololactam, thalicminine, cassythidine, acetylisocorynoline, oxynitidine, crinasiatine, zanthoxyline, and 7,9-dimethoxy-2,3-methylendioxybenzophenanthridine were found in M. cordata for the first time. The abundant alkaloids might be responsible for the bioactivity of M. cordata, which laid a foundation for the study of quality control, livestock, and clinical applications of M. cordata. {Graphical Abstract}