Photophysical studies of N-Acetyl Tryptophanamide (NATA) with urea derivatives were carried out in aqueous solution. The urea derivatives were categorised into symmetrical and unsymmetrical urea derivatives. Addition of urea derivatives result in a significant change in the absorbance maximum of NATA. Interestingly, the addition of urea and N, N’-1, 3 dimethylurea (DMU), a symmetrical urea derivative results in a fluorescence enhancement whereas an unsymmetrical urea derivative like N-butyl urea results in a fluorescence quenching. On the contrary, N-methyl urea (MU) exhibits no significant change in the fluorescence intensity, whereas N-ethyl urea (EU) results in a fluorescence enhancement to a considerable extent. The variation in excited state properties of NATA with urea derivatives are correlated to the variation in the excited state dipole moment of the indole moiety microenvironment. Various hydrogen-bonding arrangements exits between NATA and urea derivatives and this is correlated to the shift in the emission maxima towards the red region. NATA exhibits a single exponential decay and the fluorescence lifetime in aqueous solution is 3.0 ns. Addition of urea derivatives results in a significant increase in the fluorescence lifetime irrespective of the nature of the urea derivatives. Butyl Urea(BU), which displays a red shift accompanied with fluorescence quenching of NATA exhibits contrasting properties compared to other urea derivatives is established through fluorescence spectral studies.