Substrate-free electrodes are promising dry electrodes for long-term physiological electrical signal monitoring due to their ultra-thinness, conformal contact, and stable skin–electrode impedance. However, the response of substrate-free electrodes to various disturbances during electrocardiogram (ECG) monitoring and the corresponding optimization needs to be investigated. This paper investigates the specific effects of various influencing factors on skin–electrode impedance and ECG during electrocardiogram (ECG) detection. The research utilizes substrate-free poly(3,4-ethylenedioxythiophene)/poly(styrene-sulfonate) (PEDOT:PSS) electrodes. The investigation employs several methods, including skin–electrode impedance comparison, ECG waveform analysis, spectrum analysis, and signal-to-noise ratio (SNR) evaluation. To avoid the impact of physiological state differences in subjects at different times, relevant data were only compared with the same group of experiments conducted in the same period. The results demonstrate that the substrate-free conformal contact PEDOT:PSS electrode has more stable skin–electrode impedance and could obtain a more stable ECG than partial contact electrodes (the SNR of the partial contact and conformal contact electrodes are 1.2768 ± 4.0299 dB and 7.2637 ± 1.4897 dB, respectively). Furthermore, the ECG signal quality of the substrate-free conformal contact PEDOT:PSS electrode was independent of the electrode area and shape (the SNRs of the large, medium, and small electrodes are 4.0447 ± 0.4616 dB, 3.9115 ± 0.5885 dB, and 4.1556 ± 0.5557 dB, respectively; the SNRs of the circular, square, and triangular electrodes are 9.2649 ± 0.6326 dB, 9.2471 ± 0.6806 dB, and 9.1514 ± 0.6875 dB, respectively), showing high signal acquisition capability that is the same as microneedle electrodes and better than fabric electrodes. The results of clothing friction effects show that skin–electrode impedance stability was important for ECG stability, while the impedance value was not (the SNRs of friction and non-friction electrodes are 2.4128 ± 7.0784 dB and 9.2164 ± 0.6696 dB, respectively). Moreover, the skin–electrode impedance maintains stability even at a high breathing frequency, but the ECG signal fluctuates at a high breathing frequency. This experiment demonstrates that even when the skin–electrode impedance remains stable, the ECG signal can still be susceptible to interference from other factors. This study suggests that substrate-free PEDOT:PSS that could form conformal contact with the skin has higher skin–electrode impedance stability and could measure a high ECG signal even with a small electrode area, demonstrating its potential as dry ECG electrodes, but the interference from other physiological electrical signals may require better circuit design.