The use of live attenuated influenza vectors requires a balance between decreasing virulence and preserving immunogenicity. One possible approach to enhancing immunogenicity and protective efficacy of such attenuated strains is the integration of transgenes capable of activating the innate and adaptive immunity in the viral genome. The experimental study aimed to construct recombinant attenuated influenza viruses capable of expressing human IL1β and conduct comparative analysis of their replication properties, immunostimulatory activity, and safety profile in vivo. Recombinant strains with different transgene localization were generated by the reverse genetics method; their growth characteristics and transgene expression levels were assessed. Activation of the innate immunity genes in response to infection with recombinant strains was assessed in the А549 cell culture. The attenuated phenotype was confirmed in the mouse model. In the reported study, recombinant attenuated influenza viruses having the NS1 protein truncated to 124 amino acid bases and expressing human IL1β were constructed and produced. It was confirmed that recombinant strains could express the functionally active IL1β; the increase in relative expression of the innate immunity genes (RIG-I, MDA5, OAS1) and pro-inflammatory cytokines (IL6 and TNFα) in response to the infection of cells with recombinant strains compared to the empty vector NS124 was shown. Thus, the IL6 gene expression increased more than 10-fold (p < 0.0001) and that of the TNFα gene increased 3–4-fold (p < 0.0001). The attenuated phenotype of the recombinant strains produced was confirmed in in vivo experiments, which suggests preservation of the safety profile along with the enhanced immunostimulatory activity.