Gracilaria lemaneiformis, an edible alga, with various bioactivities is a traditional dish and a favorite food. In the present study, its n-hexane extract showed strong protein tyrosine phosphatase 1B inhibitory activity after screening. To understand the activity composition, nineteen compounds were identified from this extract by GC-MS. The protein tyrosine phosphatase 1B inhibitory activity of the identified compounds was further screened by means of molecular docking individually. As a result, 2,2′-methylenebis-6-(1,1-dimethylethyl)-4-methyl-phenol with the lowest binding energy of −6.70 kcal mol⁻¹ was discovered from the complex extract, and its inhibitory activity against protein tyrosine phosphatase 1B was proved in vitro. The IC₅₀ value was 53.27 ± 0.54 μM. Therefore, this compound and its source Gracilaria lemaneiformis were suggested to be utilized as a functional food with potential protein tyrosine phosphatase 1B inhibitory activity.