Salmonella enterica subsp. enterica serovar Typhimurium (S. typhimurium) is an important zoonotic pathogen with important public health significance. To understand S. typhimurium’s epidemiological characteristics in China, multi-locus sequence typing, biofilm-forming ability, antimicrobial susceptibility testing, and resistant genes of isolates from different regions and sources (human, food) were investigated. Among them, ST34 accounted for 82.4% (243/295), with ST19 ranking second (15.9%; 47/295). ST34 exhibited higher resistance levels than ST19 (p < 0.05). All colistin, carbapenem, and ciprofloxacin-resistant strains were ST34, as were most cephalosporin-resistant strains (88.9%; 32/36). Overall, 91.4% (222/243) ST34 isolates were shown to have multidrug resistance (MDR), while 53.2% (25/47) ST19 isolates were (p < 0.05). Notably, 97.8% (45/46) of the MDR-ACSSuT (resistance to Ampicillin, Chloramphenicol, Streptomycin, Sulfamethoxazole, and Tetracycline) isolates were ST34, among which 69.6% (32/46) of ST34 isolates were of human origin, while 30.4% (14/46) were derived from food (p < 0.05). Moreover, 88.48% (215/243) ST34 showed moderate to strong biofilm-forming ability compared with 10.9% (5/46) ST19 isolates (p < 0.01). This study revealed the emergence of high-level antibiotic resistance S. typhimurium ST34 with strong biofilm-forming ability, posing concerns for public health safety.