Intracellular localization of RNA is important in regulating various cellular functions. Here, we determined the intracellular localization of the ammonium transporter (AtAMT1;4) mRNA in the sperm cells of Arabidopsis pollen by atomic force microscopy (AFM). The AFM probe was modified with cone-shaped dendron molecules, and an oligonucleotide complementary to the mRNA was attached to the apex. This configuration allowed facile detection of the mRNA within a sectioned pollen cell at the single-molecule level by measuring the mRNA–oligonucleotide interaction force. The AtAMT1;4 mRNA was largely localized in the nucleus rather than the cytosol of the sperm cells.