Plants are continuously in exposure to various environmental stresses. Cold stress is a major abiotic stress that can dramatically affect plant production. Although, almond (Prunus dulcis Mill.), an important nut crop, requires chilling during winter to develop fruiting buds but early spring chilling and late spring frost may result in crop loss. In plants association of miRNAs and their target genes in response to different stresses such as cold, salt and nutrient deficiency have been reported in several investigations. Small non-protein-coding RNAs (smRNAs) present in plants and regulate their targets genes expression in a negative manner. In this research, the pdu-miR477a-3p and its target acetate/butyrate-coA ligase AAE7 expression was evaluated in two anther and ovary tissues in two cold sensitive and resistant cultivars in almond under minus two and zero degree centigrade stress levels. After RNA extraction, cDNA synthesized by Stem-Loop Reverse Transcription method and fold changes of pdu-miR477a-3p expression was measured by qRT-PCR. The results showed that in two anther and ovary tissues in Sh12 genotype the expression of Pdu-miR477a-3p is up regulated under -2 °C stress. Also there was a negative correlation between pdu-miR477a-3p and its target PduAAE7. In this study reduction of Pdu-miR477a-3p has led to its target, PduAAE7, overexpression that subsequently increase Gln level, a necessary material for plant growth and development. According to negative regulatory role of pdu-miR477a-3p on its predicted target gene, PduAAE7, it can be considered as a cold stress regulator miRNA in almond.