The quantification of microalgae cells is crucial for the treatment of ships' ballast water. However, achieving rapid detection of microalgae cells remains a substantial challenge. Here, we develop a new method for rapid and effective detection of microalgae concentration by utilizing upconversion nanoprobes (UCNPs) of NaYF4:Er3+,Tm3+. Three ligands, carboxylated methoxypolyethylene glycols with 5000 and 2000 molecular weights (mPEG-COOH-5, mPEG-COOH-2) and D-gluconic acid sodium salt (DGAS), were used to convert hydrophobic UCNPs into a hydrophilic state through modification. The results show that the mPEG-COOH-5 modified UCNPs present the highest stability in an aqueous solution. Fourier Transform Infrared Spectroscopy (FTIR) measurements reveal the presence of a significant number of -COOH functional groups on UCNPs after the mPEG-COOH-5 modification. These -COOH groups enhance the hydrophilicity and biocompatibility of UCNPs. The soluble UCNPs were directly mixed with microalgae, and the upconversion luminescence (UCL) spectra of the UCNPs were recorded immediately after thorough shaking. This greatly reduces the measurement time and could realize rapid onboard detection. In this sensing procedure, the UCNPs with red UCL functioned as energy donors, while microalgae with red absorption served as an energy acceptor. The UCL gradually diminishes with an increase in microalgae concentration based on the inner filter effect, thus establishing a relationship between UCL and microalgae concentration. The accuracy of the detection is further validated through the traditional microscope counting method. These findings pave the way for a novel rapid strategy to assess microalgae concentration using UCNPs.