Two-dimensional (2D) perovskites are attracting broad attention for their stability and wavelength tunability. However, random crystallization of sample preparation makes it difficult to obtain 2D perovskites with pure structure, especially when the number of layers is large. Herein, we prepared 2D perovskite (C8H17NH3)2(MA)n-1PbnI3n+1 with different layers (n = 1-10). For the first time, we experimentally identified the band gap energy Eg of 2D perovskite (C8H17NH3)2(MA)n-1PbnI3n+1 with layers up to 10 by investigating specific pieces of crystal with pure emission spectra using fluorescence microscopy. Intriguingly, the relationship between Eg and n perfectly fits an exponential function rather than the pure quantum confinement effect in good agreement with the theoretical calculation based on first principles. Our results suggest that the band gap of the 2D perovskite is determined not only by quantum confinement effect, but other factors including chemical components also give significant contribution.