Sunflower (Helianthus annus L.) is one of the most important oil crops in the world. Once oil extracted, sunflower meal by-product could offer a potential alternative for various food applications due to its high protein content. Derived from food protein hydrolysates, metal-binding peptides have attracted attention as bioactive compounds to prevent metal-induced oxidation and diseases. This study aimed to investigate the Ni2+-binding ability of sunflower meal protein hydrolysates and ten peptides theoretically present in sunflower proteins using IMAC, switchSENSE®, UV–vis and CD techniques. Single and sequential enzymatic treatments were applied to produce hydrolysates using Protamex® (Prot) and Protamex followed by Flavourzyme® (Prot+Flav), respectively. MS/MS analysis of enriched Ni2+-binding peptides fractions revealed different composition of His-containing peptides among hydrolysates; however, similar to the His-containing pure peptides, the Ni2+-binding ability of all the hydrolysates was almost identical in IMAC. On the contrary, switchSENSE® studies indicated that the Ni2+-binding ability of sunflower peptides does not depend only on the presence of His residues, but also on their position along the polypeptide chain and the presence of proline, suggesting that Prot hydrolysates exhibited the highest Ni2+-binding ability. UV–vis and CD data confirmed that sunflower peptides bound onto Ni2+ through nitrogen atoms from imidazole sidechain of His residues, deprotonated amide bonds and N-terminal amino group, indicating square-planar and also octahedral geometries in the formed complexes. Finally, His-containing peptides without proline could offer a suitable strategy to design metal-binding peptides from sunflower meal by-product, with the most promising motifs being LLHVT and WLH.