Sea buckthorn (Hippophae rhamnoides L.) twigs, remaining after harvesting and pruning, are an underutilized and little-explored biomass resource. This study investigated the content of serotonin in 10 sea buckthorn cultivars (‘Maria Bruvele’, ‘Botanicheskaya Lubitelskaya’, ‘Tatiana’, ‘Otto’, ‘Leikora’, ‘Duet’, ‘Clara’, ‘Lord’, ‘Eva’, ‘Tarmo’) for the first time, and for further adjustment of the extraction conditions, cultivar ‘Maria Bruvele’ was extracted by water and water/ethanol solution with 20-25, 50, 70, and 96% ethanol at different temperatures. The results showed that 50% water/ethanol solutions are the most suitable for extraction, which makes it possible to increase the yield of serotonin. The 2-year-old twigs and bark from ‘Maria Bruvele’ collected in autumn contained higher serotonin content compared to spring-collected biomass. Serotonin sequential purification allowed the serotonin content in the fraction to increase to 26%/DM. The serotonin-rich fraction showed antimicrobial activity against gram-positive and gram-negative bacteria. In tests with salivary amylase, a serotonin-rich fraction at the amount of 0.1-0.4 mg/mL of saliva, under normal physiological conditions, tended to increase amylase activity, resulting in acceleration of starch degradation to glucose. Thus, the results support further study of the serotonin fraction for the treatment of people having underweight, malnutrition, and malabsorption conditions.