We present an in-flow ultrasensitive fluorescence detection of microRNAs (miRNAs) using spectrally encoded microgels. We researched and employed a viscoelastic fluid to achieve an optimal alignment of microgels in a straight measurement channel and applied a simple and inexpensive microfluidic layout, allowing continuous fluorescence signal acquisitions with several emission wavelengths. In particular, we chose microgels endowed with fluorescent emitting molecules designed for multiplex spectral analysis of specific miRNA types. We analysed in a quasi-real-time manner circa 80 microgel particles a minute at sample volumes down to a few microliters, achieving a miRNA detection limit of 202 fM in microfluidic flow conditions. Such performance opens up new routes for biosensing applications of particles within microfluidic devices.