This study investigated direct fluorescence generation from a nematic liquid crystal (NLC) NJU-LDn-4 under femtosecond laser excitation. The absorption, transmittance, excitation, and emission spectra of the NLC were assessed. The relationship between the femtosecond pump power and fluorescence intensity was analyzed, revealing a quadratic increase and indicating that two-photon absorption (2PA) is the primary fluorescence mechanism. The LC microstructure was designed using photoalignment technology, allowing the generated fluorescence to reflect the corresponding structure. This research can establish a foundation for tunable LC microstructured fluorescence, with potential applications in fluorescence microscopy and optoelectronics.