Regulation of ethylene biosynthesis or action has an important effect on volatiles production in tomato (Lycopersicon esculentum) fruits. To understand the role of ethylene in the biosynthesis of fatty acid-derived aroma volatiles in tomato, we used Lichun tomato from a transgenic line with strictly suppression of ethylene biosynthesis (antisense LeACS2 tomato) and its wild type background line. This study was focused on the levels of the precursor substrates, activities and transcriptional levels of aroma volatile-related enzymes, including lipoxygenase (LOX), hydroperoxide lyase (HPL) and alcohol dehydrogenase (ADH). We also investigated the different abilities of converting the precursor substrates to aroma volatiles in ethylene suppressed transgenic and wild-type (WT) tomato fruits. Our results showed that the contents of endogenous linoleic and linolenic acid in tomato fruits were ethylene depended. Suppression of ethylene biosynthesis increased the content of endogenous linolenic acid in Lichun tomato fruit and then declined the ratio of linoleic /linolenic acid. Exogenous ethylene changed the value of linoleic acid /linolenic acid in antisense LeACS2 (ACS) tomato fruit to the similar level of WT. During the ripening of wild type Lichun tomato fruit, LOX activity was ethylene and development dependent. Suppression of ethylene biosynthesis did not inhibit the transcriptional expression of LoxC gene. And the HPL and ADH activities were partial ethylene-dependent during the ripening of wild type Lichun tomato fruit. Moreover, suppression of ethylene biosynthesis also affected the bioconversion of unsaturated-fatty acid precursors to C6 aldehydes and C6 alcohols. All these results indicated that ethylene had complicated effects on the biosynthesis of fatty acid-derived armoa volatiles by affecting the precursor’s content, enzyme activities, enzyme expression and the substrate utilization.