MicroRNA (miRNA) is a promising new type of biomarkers but at a low fM level and hard to be analyzed. Herein proposed is an innovated surface plasmon resonance imaging (SPRi) method merged with a novel in-plane and vertical signal amplification strategy, that is, orthogonal signal amplification to enable a direct determination of sub-fM miRNA-15a (a multiple tumor diagnostic biomarker). The core idea is to add more mass on a target sample spot first along the surficial direction, then upward from the surface. In detection of miRNA, this was realized by coupling a miRNA-initiated surficial cyclic DNA-DNA hybridization reaction with a DNA-initiated upward cyclic polymerization reaction. A perfect SPRi sensing chip with isolated gold islands bordered by hydrophobic CYTOP was fabricated and used to obtain high-quality chip with low fabrication difficulty. As a result, SPRi contrast largely increases, able to reach a limit of detection and limit of quantification down to 0.56 and 5fM for miRNA-15a, about 107-fold improvement of sensitivity compared with a common SPRi detection. The method could quantify standard miRNA-15a spiked in human serum with an ideal recovery ranging from 98.6% to 104.9% and was validated to be applicable to the direct determination of miRNA-15a in healthy and cancer human serums. The orderly and controllable in situ sensitizing strategy is powerful and readily extendable to detection of other miRNAs.