This paper proposes a method to estimate the bispectral characteristics of fluorescent objects by using multispectral imaging data. We suppose that an image acquisition system allows multiple illuminant projections to the object surface and multiple response channels in the visible range. The Donaldson matrix, representing the bispectral characteristics, is modeled as a two-dimensional array with the excitation range (350, 700 nm) and the reflection and emission ranges (400, 700 nm). The outputs of a multispectral imaging system are described using the spectral sensitivities of a camera and the spectral functions of reflectance, emission, and excitation. The problem of estimating the spectral functions is formulated as a least-squares problem to minimize the residual error of the observations and the roughness of the spectral functions. We assume that the spectral functions are represented by a limited number of basis functions. We utilize the fact that a fluorescent object usually contains a single fluorescent material (fluorophore). The spectral reflectance and the emission spectrum are estimated using the basis functions, whereas the excitation spectrum is estimated using a physical model between excitation and reflection. An iterative algorithm is developed to obtain the optimal estimates of the three spectral functions of reflectance, emission, and excitation. The performance of the proposed method is examined in simulation experiments and compared with the other methods.