The aim of this study was to investigate the alkaloids in Plumula nelumbinis collected in the Xiangtan, Hunan province and to compare the antioxidant activity of total alkaloids fraction from Plumula nelumbinis collected from thirteen different locations in China. By comparing the retention times, mass spectrometry fragmentation ions, molecular formulas and molecular weights of the components with those of standards and reference compounds, a total of thirty alkaloids belonging to the benzylisoquinoline, bisbenzylisoquinoline, aporphine, proaporphine, isoquinolines alkaloid families were identified based on a sensitive and efficient ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry method. Six alkaloids were detected for the first time in Nelumbo and fourteen alkaloids were detected for the first time in Plumula nelumbinis. Two compounds, which named as isococlaurine-5′-O-pentoside and coclaurine-5′-O-pentoside, were characterized and identified as new compounds. The antioxidant activity of the total alkaloids collected from thirteen locations was determined by three standard assays. It was shown that Anhuibozhou province produces plants with the strongest antioxidant activity measured in the three assays, and was followed by Yunnanwenshan. Shandongweihu has the strongest oxygen radical absorbance capacity, Zhejianghangzhou produces plants with the strongest ferric reducing ability and Jiangxishicheng plants have the strongest radical-scavenging activity. By and large, a positive correlation between the alkaloid content and antioxidant activity was found. Plumula nelumbinis samples from different habitats that have antioxidant activities may be alternatives to synthetic antioxidants in the food industry.