Abstract: Lacticaseibacillus rhamnosus is one of the most researched and commercially utilized strains in recent years due to its excellent probiotic properties. In the present study, the in vitro probiotic properties of Lacticaseibacillus rhamnosus Z23, which was isolated from Hurunge, were characterized, and in vitro and in vivo safety evaluations were conducted. Finally, the whole genome sequence of the strain was determined, and the probiotic properties of the strain were explored in depth by combining genotypic and phenotypic methods. The results showed that the survival rates of strain Z23 in a pH2.5 acidic environment and a 0.5% bile salt environment for 3 hours were 85.42% and 84.04%, respectively. The survival rates in simulated artificial gastric fluid for 3 hours and simulated artificial intestinal fluid for 4 hours were 90.19% and 85.96%, respectively. The hydrophobicity to xylene, ethyl acetate, and n-hexadecane was 45.08%, 19.33%, and 33.10%, respectively. The adherence rate to human colon carcinoma cells (Caco-2) after 3 hours was 5.20%, and the auto-aggregation rate of strain Z23 after 5 hours was 74.63%, all of which were better than those of LGG. The co-aggregation rates against four common human pathogenic bacteria were above 36%. Moreover, it exhibited an inhibitory effect on seven common human pathogenic bacteria. Strain Z23 was found to have a strong antioxidant capacity, especially in the bacterial suspension and fermentation supernatant, where higher scavenging rates of hydroxyl radicals and superoxide anion radicals than those of LGG were observed. However, lower scavenging rates of DPPH radicals and total antioxidant capacity (T-AOC) compared to LGG were detected. The antioxidant capacity of strain Z23 was mainly concentrated in the bacterial suspension and the fermentation supernatant. In vitro safety tests showed that no hemolytic activity and no amino acid decarboxylase activity were exhibited by strain Z23. Sensitivity to five antibiotics, including clindamycin, tetracycline, chloramphenicol, erythromycin, and ampicillin was observed. The results of the oral acute toxicity test in mice indicated that it was safe and non-toxic. The whole genome sequence size of strain Z23 was 3006999 bp, with an average GC content of 46.65%, containing a chromosome of 2960559 bp and a plasmid of 46440 bp. The whole genome contained 2800 protein-coding genes (CDSs). A total of two clusters of secondary metabolite synthesis genes (T3PKS and bacteriocin) were identified. It also had several genes related to acid tolerance, bile salt tolerance, and antioxidant properties, and it did not express pathogenic genes. In summary, strain Z23 is a strain with excellent probiotic properties and safety, which can provide strain resources for its future application research, development, and utilization.