Kiwifruit seeds are usually discarded as waste in the kiwifruit processing industry. However, kiwifruit seeds are rich in oil, which implies their value as a potential resource. Therefore, three-phase partitioning (TPP) using ammonium sulfate and n-butanol has been developed to extract kiwifruit seed oil (KSO) from discarded kiwifruit seeds. The factors influencing the extraction process have been investigated. The highest extraction yield of 36.06% for KSO was obtained under the following conditions: an extraction time of 18 h, a liquid-solid ratio of 4:1, an amount of ammonium sulfate of 28.5 wt%, and a phase ratio (top phase/bottom phase) of 1/1. The composition of obtained KSO was analyzed by gas chromatography-mass spectrometry (GC-MS), and the results indicated the high content of α-linolenic acid. This simple and low-cost method can be used as an efficient approach for utilizing the value of kiwifruit seeds.