Myoglobin (Mb) is a critical cytoplasmic heme protein and one of the earliest detectable biomarkers following acute myocardial infarction (MI). The rapid and precise detection of serum Mb holds significant clinical value. This study capitalizes on the advantages of a split aptamer (Apt) sequence, which is short and effectively minimizes false-positive signals caused by the secondary structure of the probe. It constructs a "sandwich"-structured liquid crystal biosensor with an "Apt-target molecule-Apt" configuration for the label-free detection of serum Mb. Mb Apt fragments were immobilized on the surface of a glass substrate as capture probes (CP) by using triethoxybutanal silane (TEA) linker molecules. When Mb is present, the other Apt fragment, the signal probe (SP), specifically binds and reassociates with the CP, restoring the original stem-loop structure of Apt, thereby inducing a change in the alignment of 5CB liquid crystal molecules. This change leads to variations in the brightness and color of the optical signal in the polarizing microscope image, facilitating the quantitative detection of serum Mb. This method is capable of detecting Mb concentrations ranging from 0.05 to 130 ng/mL, with a minimum detection limit of 0.045 ng/mL. This "Apt-target molecule-Apt" sandwich liquid crystal biosensing method offers several advantages, including label-free detection, operational simplicity, and high specificity. It demonstrates significant potential for the rapid detection of serum biomarkers.