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Alfin-like proteins (ALs) form a plant-specific transcription factor (TF) gene family involved in the regulation of plant growth and development, and abiotic stress response. In this study, 30 ALs were identified in <i>Brassica napus</i> ecotype ‘Zhongshuang 11’ genome (BnaALs), and unevenly distributed on 15 chromosomes. Structural characteristic analysis showed that all of the BnaALs contained two highly conserved domains: the N terminal DUF3594 domain and the C-terminal PHD-finger domain. The BnaALs were classified into four groups (Group I-IV), supported by conserved intron–exon and protein motif structures in each group. The allopolyploid event between <i>B. oleracea</i> and <i>B. rapa</i> ancestors and the small-scale duplication events in <i>B. napus</i> both contributed to the large <i>BnaALs</i> expansion. The promoter regions of <i>BnaALs</i> contained multiple abiotic stress <i>cis</i>-elements. The <i>BnaALs</i> in I-IV groups were mainly expressed in cotyledon, petal, root, silique, and seed tissues, and the duplicated gene pairs shared highly similar expression patterns. RNA-seq and RT-qPCR analysis showed that <i>BnaALs</i> were obviously induced by low nitrogen (LN) and low phosphorus (LP) treatments in roots. Overexpressing <i>BnaAL02</i> and <i>BnaAL28</i> in <i>Arabidopsis</i> demonstrated their functions in response to LN and LP stresses. <i>BnaAL28</i> enhanced primary roots’ (PRs) length and lateral roots’ (LRs) number under LP and LN conditions, where <i>BnaAL02</i> can inhibit LR numbers under the two conditions. They can promote root hair (RH) elongation under LP conditions; however, they suppressed RH elongation under LN conditions. Our result provides new insight into the functional dissection of this family in response to nutrient stresses in plants.