Phytohormones are special small molecules that play important role in plant growth and development at trace levels. Quantification of multiple phytohormones will be great helpful for researches about cross-talks that plant hormones regulate the responses of plants against both biotic and abiotic stresses by means of synergistic or antagonistic interactions. In the current study, we developed a method for profiling of phytohormones in one small sample, including indole-3-acetic acid, abscisic acid, jasmonic acid, gibberellins, cytokinins and brassinosteroids. These phytohormones mentioned above were firstly purified and separated by sequential magnetic solid-phase extraction (MSPE) and then analyzed by ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Under the optimized extraction conditions, good linearity was obtained with correlation coefficients (r) ranging from 0.9961 to 0.9998. The limits of detection (LODs, S/N = 3) were ranged from 0.45 to 126.1 pg mL−1. The recoveries were between 85.0% and 116.2%. The relative standard deviations (RSDs) were ranged from 2.7% to 16.1%. With the proposed strategy, 23 phytohormones could be purified and analyzed from a single plant extract. Finally, 16 phytohormones could be detected in 100 mg (fresh weight) flower of Brassica napus L., including IAA, ABA, JA, 4 GAs, 3 BRs and 6 CKs with the concentration ranged from 0.09 to 305.23 ng g−1.