This study investigated the possibility of using amaranth protein isolate (API) as a plant‐based substitute for egg yolk (EY) in the preparation of low‐fat mayonnaise (LFM). The alkali extraction/acidic precipitation method was used to isolate amaranth protein; its functional properties were then studied. The results showed that besides its great water and oil absorption capacities, API had better emulsifying capacity and significantly higher (p < .05) emulsion stability at pH 2.0 than alkali pH values. Five mayonnaise samples with different API/EY combination ratios (%) (i.e., 0/0.75, 0.25/0.5, 0.375/0.375, 0.5/0.25, and 0.75/0) were prepared. The color, emulsion stability (ES), freeze–thaw stability (FTS), droplet size, structure, rheology, and sensory properties of samples were examined. API replacement showed no adverse effects on the L* value, ES, and sensory attributes (p > .05). Low API concentrations (0.25% and 0.375%) significantly (p < .05) increased the droplet size and decreased the FTS of LFM emulsion. High API concentrations (0.5% and 0.75%) had no significant effect (p > .05) on droplet size and formed emulsions with more tightly packed oil droplets. The Cross model was chosen best to describe the flow behavior of LFM samples (R2 = 0.99). The sample with 0.75% API had significantly (p < .05) the highest values of ηo (zero‐shear viscosity) and λ (relaxation time), indicating greater interaction between the emulsion particles. All samples showed a weak gel structure (G' > G"). In conclusion, API can be considered an appropriate substitute for EY in LFM production, which can benefit human health and offer a new strategy for preparing vegan products.