Δ6 desaturase from oleaginous Mortierella alpina (MaFADS6) is a key enzyme in biosynthesis of ω3/ω6 PUFAs, whose substrate preference directly determines the fatty acid metabolic flux. There are two isozymes:FADS6-I and FADS6-Ⅱ in the genome of Mortierella alpina ATCC 32222. In most species, FADS6-I has been widely researched, but FADS6-Ⅱ is rarely reported. The substrate preference of Δ6-Ⅱ FADS6 was analyzed. The heterologous expression vector for FADS6-Ⅱ was constructed under the skeleton of plasmid pYES2/NT C and expressed in Saccharomyces cerevisiae. By adding exogenous substrate, the preference of Δ 6Ⅱ FADS6 was acquired. The results showed that when a single substrate was added (0.5 mmol/L linoleic acid:2Δ9,12] or 0.5 mmol/L α-linolenic acid:3Δ9,12,15]), respectively, only LA can be converted by Δ6-Ⅱ FADS6 and its conversion rate was 42.94%. When LA and ALA were added at the same time (0.25 mmol/L LA+0.25 mmol/L ALA), 37.12% LA was converted whereas no product (stearidonic acid, SDA) from ALA was determined. It will provide theoretical evidence for the function of Δ6-Ⅱ desaturase in M. alpina.