We report a novel method for estimating fluorescence impulse response function (fIRF) from noise-corrupted time-domain fluorescence measurements of biological tissue. This method is based on the use of high-order Laguerre basis functions and a constrained least-squares approach that addresses the problem of overfitting due to increased model complexity. The new method was extensively evaluated on fluorescence data from simulation, fluorescent standard dyes, ex vivo tissue samples of atherosclerotic plaques and in vivo oral carcinoma. Current results demonstrate that this method allows for rapid and accurate deconvolution of multiple channel fluorescence decays without adaptively adjusting the Laguerre scale parameter. The appropriate choice of the scale parameter is essential for accurate estimation of the fIRF. The method described here is anticipated to play an important role in the development of computational techniques for real-time analysis of time-resolved fluorescence data from biological tissues and to support the advancement of fluorescence lifetime instrumentation for biomedical diagnostics by providing a means for on-line robust analysis of fluorescence decay.