An extensive protein hydrolyzate was produced starting from sunflower defatted seed meal, an abundant by-product of biodiesel chain oil extraction. Two-step enzymatic hydrolysis was carried out using sequentially Alcalase-Flavourzyme enzymes and a high quality hydrolyzate was produced with an interesting composition in terms of nutrient, amino acid, and peptide content for application in conventional and organic agriculture. The biostimulant properties of the hydrolyzate were investigated by carrying out auxin and gibberellin-like activity assays in Petri dishes on garden cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) seedlings and by conducting experiments in pot on maize plants. The sunflower hydrolyzate showed auxin-like, but no gibberellin-like activity and interesting effects on plant root elongation suggesting a potential use of the product as an effective biostimulant in the agricultural field.