To establish spectrophotometry as a method for estimating cell number and biomass of microalgal cultures, the relationships between optical density (OD), cell number, and dry weight (DW) were investigated for four familiar algae, Chlorella sp. XQ-20044, Scenedesmus sp. SS-200716, Chlorococcum sp. and Spirulina sp. CH-164. Algal suspensions with different cell concentrations were obtained by serial dilution of cultures in exponential growth, and their ODs determined by scanning at wavelengths from 350 nm to 800 nm. The cell numbers were determined by cell counting, and biomass (as DW) was determined by weighing. Further, the ODs of the autotrophic batch cultures were scanned during the growth process, and correlated with cell numbers and DWs of the same samples. The results were as follows: for the four microalgae, cell numbers and biomass-dry weight did not change linearly with the changes in OD at all the wavelengths examined. However, the relationships could be approximately described by a linear regression equation (R² > 0.98) at 435 nm, and were well described by binomial regression equations (R² > 0.99) at other wavelengths. In continuous and semi-continuous cultures, the OD at 435 nm was suitable for estimating cell number and biomass; however, in autotrophic batch cultures an increase of the DW to OD ratio was observed during culture growth for all four algae. The ratio (DW/OD) was 0.19 to 0.44 g/L for Chlorella, 0.36 to 0.53 g/L for Scenedesmus, 0.48 to 0.75 g/L for Chlorococcum and 0.46 to 0.74 g/L for Spirulina. Thus, spectrophotometry was not suitable for estimating biomass-DW in batch culture. This study should help guide the proper application of spectrophotometry in microalgae culture.