Developing probes for the simultaneous detection of multiple tumor-associated mRNAs is beneficial for the precise diagnosis and early therapy of cancer. In this work, we prepared two COF-DNA bicolor probes at room temperature and freezing conditions and evaluated their performances in simultaneous imaging of intracellular tumor-associated mRNAs. By loading dye-labeled survivin- and TK1-mRNA recognition sequences on porphyrin COF NPs, nucleic acid-specific "off-on" nanoprobes were obtained. The nanoprobe prepared by the freezing method exhibits higher ssDNA loading density and better fluorescence quenching efficiency. Moreover, its signal-to-noise ratio is significantly higher than that prepared at room temperature, and the target recognition effect was unaffected. Significantly, the freezing-method-prepared nanoprobe has higher signal intensities in target-overexpressed cells compared to the room-temperature-prepared probe, while their signals in cells with low target expression are similar. Thus, the freezing-method-prepared nanoprobe is a promising tool for improved cancer diagnostic imaging. This work can offer new insights into the exploration of high-performance COF-based nanoprobes for multiple biomarker detection.