Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.