Harmful algal blooms (HABs) are common disastrous ecological anomalies in coastal waters. An effective algae monitoring approach is important for natural disaster warning and environmental governance. However, conducting rapid and sensitive detection of multiple algae is still challenging. Here, we designed an ultrasensitive, rapid and portable double-layer microfluidic biochip for the simultaneous quantitative detection of six species of algae. Specific DNA probes based on the 18S ribosomal DNA (18S rDNA) gene fragments of HABs were designed and labeled with the fluorescent molecule cyanine-3 (Cy3). The biochip had multiple graphene oxide (GO) nanosheets-based reaction units, in which GO nanosheets were applied to transfer target DNA to the fluorescence signal through a photoluminescence detection system. The entire detection process of multiple algae was completed within 45 min with the linear range of fluorescence recovery of 0.1 fM–100 nM, and the detection limit reached 108 aM. The proposed approach has a simple detection process and high detection performance and is feasible to conduct accurate detection with matched portable detection equipment. It will have promising applications in marine natural disaster monitoring and environmental care.