Stroke, a leading cause of death and adult disability, necessitates urgent advancements in prevention and treatment due to the lack of effective interventions. MicroRNAs (miRNAs), small noncoding RNAs, have emerged as crucial regulators in stroke pathology, influencing neuron cells through the blood-brain barrier. Our study integrated bioinformatics and experimental approaches to explore the potential of miRNAs as therapeutic targets and biomarkers for stroke. Analyzing RNA datasets, we identified 16 miRNAs and 382 mRNAs, with miR-3135b standing out as a key regulator. Utilizing bioinformatics tools, we predicted transcription factors and target genes, revealing the nuclear factor kappa B (NF-κB)/I-kappaB kinase beta (IKKβ) pathway's potential involvement. Experimental validation in a cerebral ischemia injury model demonstrated that intranasal administration of miR-3135b-agomir reversed protein levels of IKKβ and p65, inhibited the NF-κB pathway, and alleviated neuroinflammation. Behavioral assessments, molecular analyses, and immunofluorescence studies supported the therapeutic impact of miR-3135b, reducing infarct volume, enhancing neuronal regeneration, and mitigating inflammatory responses. Our findings underscore miR-3135b's promise as a therapeutic target in ischemic brain injury and advocate for further investigations into its relationship with neuroinflammation.