Lipoxygenase (EC1.13.11.12, LOX) has been potentially used in the food industry for food quality improvement. However, the low activity, poor thermal stability, narrow range of pH stability, as well as undesirable isoenzymes and off-flavors, have hampered the application of current commercial LOX. In this study, a putative mini-lipoxygenase gene from cyanobacteria,Nostoc sphaeroides(NsLOX), was cloned and expressed inE. coliBL21. NsLOX displayed only 26.62% structural identity with the reported LOX fromCyanothecesp., indicating it as a novel LOX. The purified NsLOX showed the maximum activity at pH 8.0 and 15 °C, with superior stability at a pH range from 6.0 to 13.0, retaining about 40% activity at 40 °C for 90 min. Notably, NsLOX exhibited the highest specific activity of 78,080 U/mg towards linoleic acid (LA), and the kinetic parameters—Km,kcat, andkcat/Km—attain values of 19.46 μM, 9199.75 s−1, and 473.85 μM−1s−1, respectively. Moreover, the activity of NsLOX was obviously activated by Ca2+, but it was completely inhibited by Zn2+and Cu2+. Finally, NsLOX was supplied in steamed bread and contributed even better improved bread quality than the commercial LOX. These results suggest NsLOX as a promising substitute of current commercial LOX for application in the food industry.