10-Hydroxy-12-octadecenoic acid(10-HOE)， one microbial hydroxy unsaturated fatty acid， was recently reported to have the bioactivities of the anti-fungi， anti-inflammatory， and anti-allergic effects. It has the potential to be used in food， pharmaceutical， chemical， and other industries. Thus， the biosynthesis of 10-HOE is worthy of investigation. In this work， 10-HOE was biosynthesized using the resting cell system with linoleic acid as the substrate and the recombinant Escherichia coli as the catalyst. After the single-factor experiment， the optimum conditions for the 10-HOE biosynthesis using resting cells were obtained， eg. 0.25 mmol/L flavin adenine dinucleotide (FAD)， 2% hexane， 15 mmol/L sodium chloride， 20 g/L linoleic acid， and 200 g/L cells at pH 7.0. The mixture was incubated at 37 °C and 200 r/min after a thorough mixing with a vortex mixer， and a conversion yield of (73.1±0.9)% with the 10-HOE concentration of (16.3±0.2) g/L was achieved at 24 h. Within our knowledge， this is the highest concentration for the10-HOE biosynthesis. This work provided one method of the 10-HOE biosynthesis， which might be helpful for the industrial production.