A novel alkynyl substituted N-aryl-l,8-naphthalimide Ⅱ (C20HiiNO2) was synthesi-zed for the discovery and evaluation of new fluorescence probes, and was characterized by X-ray crystal diffraction. It crystallizes in monoclinic, space group P21/n with a = 15.898(3), b = 5.0102(10), c= 17.962(4) , β = 92.97(3)°, V= 1428.7(5) A3, Z = 4, Mr = 297.30, Dc= 1.382 g/cm3, F(000) = 616, μ = 0.090 mm-1, S = 1.011, the final R = 0.0542 and wR = 0.1204 for 1669 observed reflections withⅠ>2σ(Ⅰ) and 212 variable parameters. Hydrogen-bounding and π-stacking interactions were discussed. The influence of acetylene group on the fluorescence properties was also investigated. In comparison with N-phenyl-l,8-naphthalimide Ⅰ (C18H11NO2), few new structure characters and fluorescence properties of the title compound have been found.