Nigella glandulifera Freyn, an annual plant species of the family Ranunculaceae, served as a traditional Uigur medicine and food. Many researchers have reported that Nigella sativa L., which is the same genus with it, has an antidiabetic effect, the seeds oil was an activity component used as a kind of function food. In the present study, the difference of oil extract between n-hexane and petroleum ether respectively from the seeds of Nigella glandulifera Freyn were analyzed by gas chromatography-mass spectrometry, all of them can inhibit protein tyrosine phosphatase 1B (PTP1B) with half maximal inhibitory concentration values of 33.15 ± 0.77 and 18.50 ± 0.52 μg/mL, respectively. They have different binding sites with PTP1B. Lastly, inhibited ability of PTP1B and that of TCPTP were compared; all of them have shown some selectivity.