This study presents a procedure for preconcentration of Cr(III), Cd(II) and Mn(II) from water and biological samples using Penicillium digitatum immobilized on pumice stone. Optimum conditions such as pH, flow rate were evaluated. The recoveries of Cr(III), Cd(II) and Mn(II) under optimum conditions were found to be 98 +/- 2%, 100 +/- 2%, and 97 +/- 2%, respectively, at a 95% confidence level. Detection limits were 2.0, 1.6 and 1.5 ng/mL for Cr(III), Cd(II) and Mn(II), respectively. The proposed procedure was successfully applied for the determination of chromium, cadmium and manganese in dam water, spring water and fish (Carp) samples. The accuracy was evaluated through the analysis of the certified standard reference fish tissue samples (IAEA-407) and spiked fish and water samples.